以電子方式 呈送 之序列表 之 參考
本申請案以引用的方式併入隨同此申請案呈送之以電腦可讀形式(CRF)之序列表，其為創建於2014年10月1日之標題為「Ticagrelor_SeqList_ST25.TXT」的文字檔案及係33,900個位元組大小。
在繼續更詳細描述本發明之前，應瞭解本發明並不限於特殊組合物或方法步驟，由於此可變化。須注意，除非本文清楚地另作規定，否則用於本說明書及隨附申請專利範圍中之單數形式「一」、「一個」及「該」包括複數指示物。
除非另外定義，否則本文中使用的所有技術及科學術語具有與如熟習本發明相關技術者通常所瞭解相同的含義。例如，Concise Dictionary of Biomedicine and Molecular Biology，Juo, Pei-Show，第2版，2002年，CRC Press；The Dictionary of Cell and Molecular Biology，第3版，1999年，Academic Press；及Oxford Dictionary Of Biochemistry And Molecular Biology，修訂版，2000年，Oxford University Press，提供熟習此項技術者本發明中所使用許多術語之一般辭典。
本文胺基酸可引用由IUPAC-IUB生物化學命名委員會建議之其通常熟知之三字符號或單字符號。同樣，核苷酸可引用其通常可接受之單字編碼。
除非另作說明，否則抗體之可變域、互補決定區(CDR)及框架區(FR)中胺基酸之編號，遵循Kabat定義，如由Kabat等人，Sequences of Proteins of Immunological Interest，第五版，Public Health Service, National Institutes of Health, Bethesda, MD.(1991)所陳述。使用此編號系統，實際直鏈胺基酸序列可含有對應縮短、或***可變域之FR或CDR的更少或額外胺基酸。例如，重鏈可變域可包括在H2之殘基52後之單一胺基酸***(根據Kabat之殘基52a)及在重鏈FR殘基82後之***殘基(例如，根據Kabat之殘基82a、82b、及82c，等等)。給定抗體之殘基之Kabat編號可由在具有「標準」Kabat編號序列之抗體序列同源區對準決定。框架殘基之最大對準往往需要在編號系統中***「間隔」殘基以用於Fv域。此外，由於種間或等位基因差異，在任何給定Kabat位點編號之特定個別殘基之識別可從抗體鏈至抗體鏈變化。
如本文使用，術語「抗體」及「抗體等」，亦稱為免疫球蛋白，包含單株抗體(包括全長單株抗體)、多株抗體、由至少兩個不同抗原決定基結合片段形成之多特異性抗體(例如，多特異性抗體，例如PCT公開案WO2009018386、PCT申請案第PCT/US2012/045229號，其全文以引用的方式併入本文中)、biMab、人類抗體、人源化抗體、駱駝化抗體、單鏈Fv(scFv)、單鏈抗體、單域抗體、域抗體、Fab片段、F(ab')2
片段、顯示期望生物活性之抗體片段(例如，抗原結合部分)、二硫鍵鍵結之Fvs(dsFv)、及抗獨特型(anti-Id)抗體(包括例如對本發明抗體之anti-Id抗體)、內抗體、及任何上述之抗原決定基結合片段。在本文提供之特定實施例中，抗體係關於抗體之活性結合片段，即，含有至少一個抗原結合位點之分子，諸如，例如scFv及Fab。抗體亦包括與抗體或其部分融合之肽，諸如與Fc域融合之蛋白質。免疫球蛋白分子可具有任何同型物(例如，IgG、IgE、IgM、IgD、IgA及IgY)、子同型物(例如，IgG1、IgG2、IgG3、IgG4、IgA1及IgA2)或同種異型(例如，Gm，例如，G1m(f、z、a或x)、G2m(n)、G3m(g、b、或c)、Am、Em、及Km(1、2或3))。抗體可衍生自任何哺乳動物(包括但不限於人類、猴、豬、馬、兔、狗、貓、小鼠、等等)、或其他動物諸如鳥(例如雞)。
如本文使用，C1
-C6
烷基指具有一至六個碳原子之直鏈及分支鏈烷基，及包括甲基、乙基、丙基、正丁基、異丁基、戊基、異戊基、新戊基、及己基。
如本文使用，C1
-C6
烷氧基指如上文表明在基團中具有一個氧之烷基。在一些實施例中，該氧原子位於將取代基附接至核心結構(即，環結構)之位置。
如本文使用，C1
-C6
烷硫基指如上文表明在基團中具有一個硫之烷基。在一些實施例中，該硫原子位於將該取代基附接至核心結構(即，環結構)之位置。
如本文使用，C1
-C6
烷醇指如上文表明在該取代基結構末端具有羥基之烷基。
如本文使用，C3
-C6
環烷基指環丙基、環丁基、環戊基、及環己基。
如本文使用，「經取代」之C3
-C6
環烷基及C1
-C6
烷基指上文討論之在至少一碳原子上由亦經1至3個鹵素原子取代之芳基取代的烷基及環烷基。
如本文使用，「替格瑞洛」指可逆P2Y12
抑制劑((1S,2S,3R,5S)-3-[7-{[(1R,2S)-2-(3,4-二氟苯基)環丙基]胺基}-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-3-基]-5-(2-羥基乙氧基)環戊烷-1,2-二醇)及具有以下化學結構：。
如本文使用，「替格瑞洛活性代謝物」或「TAM」指替格瑞洛之主要活性代謝物，亦稱為AR-C124910XX，一種可逆P2Y12
抑制劑及具有以下化學結構：。
如本文使用，「替格瑞洛非活性代謝物」或「TIM」指替格瑞洛之非活性代謝物，亦稱為AR-C133913XX，及具有以下化學結構：。抗體
一般意義上，本發明提供結合式(Ia
)之環戊基***并嘧啶化合物的新穎抗體：
其中
R1
係選自由C1
-C6
烷氧基及C1
-C6
烷硫基組成之群；
R2
係選自由H、C1
-C6
烷基、經取代之C1
-C6
烷基、C3
-C6
環烷基、及經取代之C3
-C6
環烷基組成之群；且
R3
係選自由H、C1
-C6
烷基、C1
-C6
烷氧基、及C1
-C6
烷醇組成之群。
在特定實施例中，該抗體特異性結合選自由如下組成之群的化合物：替格瑞洛；替格瑞洛活性代謝物(TAM)；及替格瑞洛非活性代謝物(TIM)。
在特定態樣中，本發明提供一種具有任何一或數個如下特徵的結合至替格瑞洛及TAM之抗體，該等特徵包括高結合特異性、高結合親和性、快速起效、及快速抵消(例如，容許視情況延續或共投與包括替格瑞洛之治療)。
在一些實施例中，該抗體結合至替格瑞洛及中和替格瑞洛及TAM之抗血小板凝聚活性，由此恢復在替格瑞洛與TAM存在下ADP誘發之血小板凝聚。
在一些實施例中，在受試者中該抗體半衰期係約等於替格瑞洛及TAM之半衰期。在一些實施例中，該抗體之半衰期係約4至24小時(例如，4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24小時)。在一些實施例中，該抗體半衰期係約4至12小時(例如，4、5、6、7、8、9、10、11、或12小時)。
在一些實施例中，該抗體提供活性快速起效。例如，在實施例中，該抗體起效時間或中和替格瑞洛及TAM介導之血小板抑制的時間係約15至120分鐘、或約15至60分鐘。在一些實施例中，起效時間小於60分鐘。
在一些實施例中，該抗體具有提供快速活性抵消之PK/PD曲線，使得例如經投與該抗體之受試者可重新開始指定替格瑞洛治療。在一些實施例中，接受本文揭示抗體 (例如，藉由i.v.注入)之受試者可在投與該抗體後二十四小時內接受或重新開始替格瑞洛治療。
如在本文某些實施例中討論及例示，該抗體結合替格瑞洛或其代謝物及不結合至其他結構相關化合物，或可作為共治療與替格瑞洛一起投與之化合物。例如，適宜地，該抗體不抑制性選自由非諾貝特、尼伐地平、西洛他唑、布拉地新、瑞加德松、環噻嗪、賽扶寧、洛伐他汀、利奈唑胺、辛伐他汀、坎格瑞洛、潘妥拉唑、腺苷、二磷酸腺苷、三磷酸腺苷、二磷酸2-MeS腺苷、及三磷酸2-MeS腺苷組成之群的化合物之活性。
本文描述之抗體可包括僅含有抗體分子之選定部分之抗原結合片段(諸如Fab、F(ab')2
、Fab'、scFv、di-scFv、sdAb片段)及可用作診斷或治療劑。此外，可改變可變域中特異性殘基以改良抗體及抗體片段之結合特異性及/或穩定性。不直接涉及抗原結合之其他殘基可經替代以「人源化」非人類抗體之區域及降低抗體之免疫原性。
在某些態樣中，該抗體係Fab片段，例如，包括可變輕鏈(VL)、恆定輕鏈(CL)、可變重鏈(VH)、及恆定重鏈部分(CH1)的抗體Fab片段或重組產生之抗原結合片段。視情況，該Fab之輕及重鏈可經由一或數個二硫鍵，諸如，例如，經由適宜抗體鉸鏈區互連。如本文描述，Fab結合至環戊基***并嘧啶類口服活性劑化合物之抗原決定基。在一些實施例中，Fab結合至替格瑞洛或其代謝物。
在某些態樣中，Fab可衍生自或基於抗體序列，諸如習知鼠類、人源化或人類抗體。在某些態樣中，Fab可衍生自或基於一或數個scFv，諸如從庫篩選及衍生之scFv。在此等實施例中，衍生自或基於習知抗體或scFv之序列之Fab保留習知抗體之一或數個功能活性(例如，保留至少80%或更多(80%、85%、90%、95%、97%、98%、99%或100%)功能活性)。例如，在某些態樣中，Fab保留抗原(例如，替格瑞洛)親和性、抑制活性、及/或抗體或scFv之選擇性中之一或多者。
儘管Fab片段可包括結合至環戊基***并嘧啶之抗原決定基之序列，在某些實施例中，Fab結合至替格瑞洛。在一些態樣中，Fab結合至替格瑞洛活性代謝物。在某些態樣中，Fab可結合至替格瑞洛及替格瑞洛活性代謝物二者。
在一些實施例中，Fab可包括獲自結合至替格瑞洛或其活性代謝物之不同抗體的CDR區之組合。
在某些態樣中，Fab包括輕鏈部分(VL)，其包括在SEQ ID NO:7、SEQ ID NO:17、SEQ ID NO:27、SEQ ID NO:37、SEQ ID NO:47、SEQ ID NO:57、SEQ ID NO:67、及SEQ ID NO:77中任一者列出之胺基酸序列。在其他實施例中，Fab包括輕鏈部分，其包括在SEQ ID NO:57、SEQ ID NO:67、及SEQ ID NO:77中任一者列出之胺基酸序列。在某些態樣中，Fab包括重鏈部分(VH)，其包括在SEQ ID NO:2、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:32、SEQ ID NO:42、SEQ ID NO:52、SEQ ID NO:62、及SEQ ID NO:72中任一者列出之胺基酸。在其他實施例中，Fab包括重鏈部分，其包括在SEQ ID NO:52、SEQ ID NO:62、及SEQ ID NO:72中任一者列出之胺基酸序列。在某些態樣中，Fab由編碼輕鏈部分(VL)之核苷酸序列及編碼重鏈部分(VH)之核苷酸序列，例如，包括在SEQ ID NO:1、SEQ ID NO:11、SEQ ID NO:21、SEQ ID NO:31、SEQ ID NO:41、SEQ ID NO:51、SEQ ID NO:61、或SEQ ID NO:71中列出之核酸序列的核苷酸序列；及包括在SEQ ID NO:6、SEQ ID NO:16、SEQ ID NO:16、SEQ ID NO:16、SEQ ID NO:16、SEQ ID NO:16、SEQ ID NO:16、或SEQ ID NO:76中列出之核酸序列的核苷酸序列編碼。
在某些態樣中，該抗體可係scFv。應瞭解，scFv涵蓋包括經由撓性多肽連接子連接至可變輕鏈域(VL)之可變重鏈域(VH)的多肽鏈。在一些態樣中，在VH與VL間之多肽連接子包括蛋白酶裂解位點。scFv之VH及VL域可衍生自相同或不同抗體。在一些態樣中，該scFv之VH或VL可包括一或數個結合至所關注靶之CDR，同時VH或VL域之剩餘部分係衍生自不同抗體或係合成。在一些態樣中，scFv包括抗體(例如，具有結合至替格瑞洛或其代謝物之結合活性的抗體)之至少一CDR。在一些態樣中，scFv包括給定抗體之至少兩個CDR。在一些態樣中，scFv包括給定抗體之至少三個CDR。在一些態樣中，scFv包括給定抗體之至少四個CDR。在一些態樣中，scFv包括給定抗體之至少五個CDR。在一些態樣中，scFv包括給定抗體之至少六個CDR。
可單獨或組合使用若干方法以改良scFv分子之穩定性。可單獨或與一或數個其他方法組合使用之一方法係改造連接scFv域之連接子之長度及/或組成以穩定scFv部分。
可單獨或與一或數個本文描述之其他方法組合使用之另一潛在方法係藉由將至少兩個胺基酸取代基引入(亦稱為修飾或突變)該scFv之VH及/或VL域以促進二硫鍵形成(例如，參見Brinkmann等人，1993，PNAS，90:7538-42；Zhu等人，1997，Prot. Sci. 6:781-8；Reiter等人，1994，Biochem. 33:5451-9；Reiter等人，1996，Nature 14: 1239-45；Luo等人，1995，J. Biochem. 118:825-31；Young等人，1995，FEBS Let. 377:135-9；Glockshuber等人，1990，Biochem. 29:1362-7)。
在某些態樣中，將一突變引入scFv之各個VH及VL域以促進scFv表現時在VH與VL域間鏈間二硫鍵形成。在另一態樣中，將該兩個突變引入該鏈之相同域。在某些態樣中，將該兩個突變引入不同鏈中。在某些態樣中，引入多對兩個突變以促進多個二硫鍵形成。在某些態樣中，引入半胱胺酸以促進二硫鍵形成。可突變為半胱胺酸之例示性胺基酸包括VH2之胺基酸43、44、45、46、47、103、104、105、及106與VL2之胺基酸42、43、44、45、46、98、99、100、及101。先前編號係基於Kabat編號，其確定僅與scFv之VH2及VL2有關(及與在全長抗體序列中胺基酸之位置無關)之位置。可突變為半胱胺酸殘基之胺基酸部分之例示性組合包括：VH44-VL100、VH105-VL43、VH105-VL42、VH44-VL101、VH106-VL43、VH104-VL43、VH44-VL99、VH45-VL98、VH46-VL98、VH103-VL43、VH103-VL44、及VH103-VL45。在一些態樣中，VH之胺基酸44及VL之胺基酸100突變為半胱胺酸。
可單獨或與一或數個本文描述之其他方法組合使用之又一潛在方法係選擇scFv域之順序。在某些態樣中，出於穩定性目的優化VH域相對於VL域之定向。在某些態樣中，scFv係在VH-連接子-VL定向。在某些態樣中，scFv係在VL-連接子-VH定向。
可單獨或與一或數個本文描述之方法組合使用之額外方法係藉由突變scFv之一或數個表面殘基引入一或數個穩定突變。在一些態樣中，一、二、三、四、五、六、或超過六個殘基在scFv之VH及/或VL域之一或二者中突變。在某些態樣中，改變僅在scFv之VH域中發生。在某些態樣中，改變僅在scFv之VL域中發生。在某些態樣中，改變在scFv之VH及VL域中均發生。可在各個域發生相同數量之改變或可在各個域中發生不同數量之改變。在某些態樣中，一或數個改變係獲自存在於未經修飾之親本scFv中之殘基的保守胺基酸取代。在其他態樣中，一或數個改變係獲自存在於未經修飾之親本scFv中之殘基的非保守胺基酸取代。當產生數個取代時，在該scFv之VH或VL域之一或二者中，各個取代獨立地為保守或非保守取代。在某些態樣中，全部取代係保守取代。在某些態樣中，全部取代係非保守。在某些態樣中，取代之至少一者係保守。在某些態樣中，取代之至少一者係非保守。
可單獨或與一或數個本文描述之額外方法組合使用之又一方法係藉由突變一或數個存在於scFv之VH及/或VL域中之殘基引入一或數個取代以匹配在已知、經篩選、及/或識別抗體之VH及/或VL域共有序列之該特定位置的最常見殘基。在某些態樣中，於scFv之VH域及/或VL域之一或二者中之一、二、三、四、五、六、或超過六個位置引入取代。可在各個域中發生相同數量之改變或在各個域中發生不同數量之改變。在某些態樣中，在序列中一或數個匹配給定共有序列之改變係獲自存在於未經修飾之VH及/或VL序列中之殘基的保守胺基酸取代。在其他態樣中，一或數個改變代表獲自存在於未經修飾之VH及/或VL序列中之殘基的非保守胺基酸取代。當產生多個取代時，在scFv之VH或VL域之一或二者中，各個取代獨立地為保守及非保守取代。在某些態樣中，全部該取代係保守取代。在某些態樣中，全部取代係非保守取代。在某些態樣中，取代之至少一者係保守。在某些態樣中，取代之至少一者係非保守。
應注意，任何描述為用於修飾或穩定scFv部分之修飾可用於修飾Fab部分。例如，可修飾Fab之可變域以改良穩定性、抗原結合及等等。此外，可修飾Fab或scFv部分以降低免疫原性。
在某些態樣中，該抗體可係包括可變輕鏈部分(VL)之scFv，該可變輕鏈部分包括在SEQ ID NO:7、SEQ ID NO:17、SEQ ID NO:27、SEQ ID NO:37、SEQ ID NO:47、SEQ ID NO:57、SEQ ID NO:67、及SEQ ID NO:77中之任一者列出之胺基酸序列。在其他實施例中，該scFv包括輕鏈部分，其包括在SEQ ID NO:57、SEQ ID NO:67、及SEQ ID NO:77中之任一者列出之胺基酸序列。在某些態樣中，scFv包括重鏈部分(VH)，其包括在SEQ ID NO:2、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:32、SEQ ID NO:42、SEQ ID NO:52、SEQ ID NO:62、及SEQ ID NO:72中之任一者列出之胺基酸。在其他實施例中，scFv包括重鏈部分，其包括在SEQ ID NO:52、SEQ ID NO:62、及SEQ ID NO:72中之任一者列出之胺基酸序列。
本文揭示之抗體可進一步包括一或數個連接子多肽。該連接子可使重鏈域及輕鏈域(scFv)互連或將抗體或其抗原結合片段連接至另一試劑，諸如標識，Fc域或等等。連接子長度及序列可變化及一般在此項技術中為吾人熟知。
可藉由增加Fc區對FcRn之結合親和性來增加包括Fc區之抗體的血清半衰期。本文使用之術語「抗體半衰期」意指為抗體分子在其投藥後之平均存活時間之量度的抗體之藥物動力學性質。抗體半衰期可表示為從患者體內(或其他哺乳動物)或其特異性隔室(例如，在血清中測量，即循環半衰期，或在其他組織中)中消除50%已知量免疫球蛋白所需之時間。半衰期可從一免疫球蛋白或一類免疫球蛋白至另一者變化。一般而言，增加抗體半衰期導致在所投與之抗體循環中之平均滯留時間(MRT)之增加。
增加半衰期可容許降低給予患者之試劑量以及降低投藥頻率。為增加抗體之血清半衰期，吾人可將補救受體結合抗原決定基併入該抗體(特定言之抗體片段)，例如，如在美國專利第5,739,277號中所描述。如本文使用，術語「補救受體結合抗原決定基」指負責增加IgG分子之體內血清半衰期之IgG分子(例如，IgG1、IgG2、IgG3、或IgG4)之Fc區的抗原決定基。或者，具有增加半衰期之本發明抗體可藉由修飾涉及Fc與FcRn受體間交互作用所確定之胺基酸殘基產生(例如，參見美國專利第6,821,505號及第7,083,784號；及WO 09/058492)。此外，可藉由在此項技術中廣泛採用之技術共軛至PEG或白蛋白增加本發明抗體之半衰期。
在本發明範疇內之抗體可藉由任何本文確定之結構及/或功能特徵進行確定。例如，可使用任何本文闡明或在此項技術中熟知之其他技術篩選抗體之特定結合特徵(例如，Koff
、KD
、IC50
、對替格瑞洛及替格瑞洛代謝物之特異性/選擇性)。標識、共軛物及部分
本發明之抗體可共軛至出於診斷及其他分析目的之標識，其中可檢測出該抗體及/或其靶。標識包括(但不限於)發色團、熒光團、熒光蛋白質、發磷光染料、串聯染料、粒子、半抗原(hapten)、酶及放射性同位素。
在某些態樣中，抗體共軛至熒光團。附接至抗體之熒光團之選擇將決定共軛抗體之吸收及熒光發射性質。可用於抗體及抗體結合配位體之熒光團標識的物理性質包括(但不限於)光譜特性(吸收、發射及斯托克斯(stokes)位移)、熒光強度、衰退期、偏光及光脫色速率、或其組合。全部此等物理性質可用於區分一熒光團與另一者，及由此容許多重分析。熒光標識之其他期望性質可包括細胞滲透性及低毒性，例如若抗體標識係在細胞或模型生物(例如，活體動物)中進行。
在某些態樣中，酶係標識及共軛至抗體。由於可達成導致分析敏感性增強之可檢測信號放大，酶係期望標識。酶本身不產生可檢測應答，但當其與適當底物接觸時用於破壞底物使得經轉化之底物產生熒光、比色或發光信號。由於在標識試劑上一種酶可導致多個底物轉化為可檢測信號，酶放大該可檢測信號。選擇酶底物以產生較佳可測量產物，例如比色分析、熒光或化學發光。此等底物係廣泛用於此項技術中及為熟習此項技術者所熟知及包括例如氧化還原酶諸如辣根過氧物酶及底物諸如3,3'-二胺基聯苯胺(DAB)；磷酸酶諸如酸性磷酸酶、鹼及底物諸如磷酸5-溴-6-氯-3-吲哚基酯(BCIP)；糖苷酶，諸如β-半乳糖苷酶、β-葡萄醣醛酸酶或β-葡萄糖苷酶及底物諸如5-溴-4-氯-3-吲哚基β-D-吡喃半乳糖苷(X-gal)；額外酶包括水解酶諸如膽鹼酯酶及肽酶、氧化酶諸如葡萄糖氧化酶及細胞色素氧化酶、及熟知其之適宜底物之還原酶。
產生化學發光之酶及其適當底物係適宜用於一些分析。此等包括(但不限於)熒光素酶及水母素之天然及重組形式。用於磷酸酶、糖苷酶及氧化酶之產生化學發光之底物諸如含有穩定二氧雜環丁烷、魯米諾、異魯米諾及吖錠酯之彼等係額外有用。
在另一態樣中，半抗原諸如生物素亦用作標識。生物素係有用，由於其可作用於酶系統以進一步放大可檢測信號，及其可用作用於出於分離目的親和性層析法之標籤。出於檢測目的，使用具有生物素親和性之酶共軛物，諸如抗生物素蛋白-HRP。隨後添加過氧化酶底物以產生可檢測信號。
半抗原亦包括激素、天然及合成藥物、污染物、過敏原、效應分子、生長因子、趨化細胞素、細胞因子、淋巴細胞活素、胺基酸、肽、化學中間產物、核苷酸及等等。
在某些態樣中，熒光蛋白可作為標識共軛至抗體。熒光蛋白之實例包括綠熒光蛋白(GFP)及藻色素蛋白及其衍生物。該熒光蛋白(特定言之藻色素蛋白)係特別適用於產生串聯染料標識之標識試劑。出於獲得較大斯托克斯位移之目的，此等串聯染料包括熒光蛋白及熒光團，其中發射光譜係從熒光蛋白吸收光譜之波長進一步位移。
在某些實施例中，該標識係放射性同位素。適宜放射性物質之實例包括(但不限於)碘(121
I、123
I、125
I、131
I)、碳(14
C)、硫(35
S)、氚(3
H)、銦(111
In、112
In、113
mIn、115
mIn)、鎝(99
Tc、99
mTc)、鉈(201
Ti)、鎵(68
Ga、67
Ga)、鈀(103
Pd)、鉬(99
Mo)、氙(135
Xe)、氟(18
F)、153
SM、177
Lu、159
Gd、149
Pm、140
La、175
Yb、166
Ho、90
Y、47
Sc、186
Re、188
Re、142
Pr、105
Rh及97
Ru。
在一些態樣中，藥物可共軛至該抗體。例如，包括scFv之抗體可共軛至用於治療心血管疾病及/或急性冠心症之藥物。
在某些特徵中，藥物及其他分子可經由位點特異性共軛靶向抗體。例如，該抗體可包括產生用於共軛反應之游離硫醇基的半胱胺酸改造域(包括改造為結合單元及/或Fc域之半胱胺酸)。在某些態樣中，該抗體經改造以併入特異性共軛位點。 編碼抗體之核酸分子
本發明提供編碼抗體或其抗原結合片段之核酸分子。本發明之一態樣提供編碼本文特別描述之任何抗體的核酸分子。核酸分子可編碼該抗體之重鏈及/或輕鏈可變區。
在一些態樣中，該抗體係Fab或scFv，其中編碼Fab或scFv之核酸部分包括編碼VL域之核苷酸序列及編碼VH之核苷酸序列，及其中編碼VL域之核苷酸序列係視情況經由編碼撓性多肽連接子之核苷酸序列連接至編碼VH域之核苷酸序列。
另一態樣提供一種經如本文描述之任一核酸分子轉形的宿主細胞。在本發明另一態樣中，提供一種包括含如本文描述之核酸分子之載體的宿主細胞。在一態樣中，該宿主細胞可包括數個載體。
本發明設計編碼任何本發明抗體，以及該抗體之輕或重鏈之核酸分子。例如，本發明設計包括編碼一或數個SEQ ID NO:2、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:32、SEQ ID NO:42、SEQ ID NO:52、SEQ ID NO:62、SEQ ID NO:72、SEQ ID NO:7、SEQ ID NO:17、SEQ ID NO:27、SEQ ID NO:37、SEQ ID NO:47、SEQ ID NO:57、SEQ ID NO:67、及SEQ ID NO:77之核苷酸序列的核酸分子。本發明進一步設計編碼進一步包括額外區(例如，Fc或經修飾之Fc)之任何本發明之抗體的核酸分子。在一些實施例中，該等核酸分子可選自一或數個SEQ ID NO:1、SEQ ID NO:6、SEQ ID NO:11、SEQ ID NO:16、SEQ ID NO:21、SEQ ID NO:26、SEQ ID NO:31、SEQ ID NO:36、SEQ ID NO:41、SEQ ID NO:46、SEQ ID NO:51、SEQ ID NO:56、SEQ ID NO:61、SEQ ID NO:66、SEQ ID NO:71、或SEQ ID NO:76。在其他實施例中，本發明提供一種載體，其包括選自一或數個SEQ ID NO:1、SEQ ID NO:6、EQ ID NO:11、SEQ ID NO:16、SEQ ID NO:21、SEQ ID NO:26、SEQ ID NO:31、SEQ ID NO:36、SEQ ID NO:41、SEQ ID NO:46、SEQ ID NO:51、SEQ ID NO:56、SEQ ID NO:61、SEQ ID NO:66、SEQ ID NO:71、或SEQ ID NO:76之核酸分子。 製備抗體、 Fab 及 scFv 之方法
本發明提供製備本文描述之抗體及其片段之方法。在一些態樣中，可藉由任何熟習此項技術者熟知之任何技術產生識別本文揭示之替格瑞洛及替格瑞洛及/或TAM之特異性抗原決定基的抗體之抗原結合片段。例如，可使用酶諸如木瓜酶(製備Fab片段)或胃蛋白酶(製備F(ab')2
片段)，藉由免疫球蛋白分子之蛋白酶裂解由抗體製備Fab及F(ab')2
片段。此外，可使用各種在此項技術中已知之噬菌體呈現方法產生如本文描述包括scFv及Fab之抗體。
一般而言，在噬菌體呈現方法中，功能抗體域係在攜帶編碼其之多核苷酸序列的噬菌體粒子表面上呈現。特定言之，編碼VH及VL域之DNA序列從動物cDNA庫(例如，淋巴組織之人類或哺乳動物cDNA庫)擴增。編碼VH及VL域之DNA藉由PCR與scFv連接子重組在一起及選殖為噬菌粒載體。該載體在大腸桿菌中電穿孔及大腸桿菌經輔助噬菌體感染。用於此等方法之噬菌體可係包括fd及M13之絲狀噬菌體及VH及VL域可以重組方式融合至噬菌體基因III或基因VIII。表現結合至替格瑞洛及/或TAM之抗原結合域之噬菌體可由抗原選擇或確定，例如使用標記抗原或結合或捕獲至固體表面或珠粒的抗原。相似地，結合至除替格瑞洛及/或TAM外或其他之抗原/半抗原的結合域可確定為取消選擇。用於製備本發明抗體之噬菌體呈現方法之實例可包括彼等在Brinkman等人，1995, J. Immunol. Methods 182:41-50；Ames等人，1995, J. Immunol. Methods 184:177-186；Kettleborough等人，1994，Eur. J. Immunol. 24:952-958；Persic等人，1997，Gene 187:9-18；Burton等人，1994，Advances in Immunology 57:191-280；PCT申請案第PCT/GB91/O1 134號；PCT 公開案第WO 90/02809號、第WO 91/10737號、第WO 92/01047號、第WO 92/18619號、第WO 93/1 1236號、第WO 95/15982號、第WO 95/20401號、及第WO97/13844號；及美國專利第5,698,426號、第5,223,409號、第5,403,484號、第5,580,717號、第5,427,908號、第5,750,753號、第5,821,047號、第5,571,698號、第5,427,908號、第5,516,637號、第5,780,225號、第5,658,727號、第5,733,743號及第5,969,108號中所揭示者；其各者之全文以引用的方式併入本文中。
如在上文參考文獻中所描述，在噬菌體選擇後，可自噬菌體單離抗體編碼區域及用於產生全抗體，包括人類抗體、或任何其他期望抗原結合片段(scFv及Fab)，及在任何期望宿主(包括哺乳動物細胞、昆蟲細胞、植物細胞、酵母、及細菌，例如，如下文描述)中表現。亦可使用在此項技術中熟知之方法諸如彼等在PCT公開案第WO 92/22324號；Mullinax等人，1992，BioTechniques 12(6):864-869；Sawai等人，1995，AJRI 34:26-34；及Better等人，1988，Science 240:1041-1043 (該等參考文獻其全文以引用的方式併入本文中)中所揭示之方法，採用重組製備Fab、Fab'及F(ab')2
片段之技術。
在某些態樣中，本文揭示之核酸以可操作方式連接至在表現構造中之一或數個調節核苷酸序列。編碼抗體輕及重鏈之核酸序列可在相同表現載體中以任何方向(例如，輕鏈在重鏈前端或相反)選殖或可在兩個不同載體中選殖。若表現係使用一個載體進行，該兩個編碼基因可具有其各自遺傳因子(例如，啟動子、RBS、引導段、停止、polyA等等)或其可由單一組遺傳因子選殖，但與順反子相連。調節核苷酸序列將一般適宜於用於表現之宿主細胞。針對各種宿主細胞之數個類型之適當表現載體及適宜調節序列係在此項技術中熟知。通常，該一或數個調節核苷酸序列可包括(但不限於)啟動子序列、引導段或信號序列、核糖體結合位點、轉錄起始及終止序列、翻譯起始及終止序列、及增強或活化序列。如在此項技術中熟知之組成型或誘導型啟動子係由本發明設計。啟動子可係天然啟動子、或組合數個啟動子因子之雜合啟動子。表現構造可存在於細胞中之附加體(諸如質體)上、或表現構造可***染色體中。
在某些態樣中，表現載體含有可選擇標記基因以容許選擇經轉形之宿主細胞。可選擇標記基因係在此項技術中為吾人熟知及可隨使用之宿主細胞變化。在某些態樣中，本發明係關於包括編碼多肽及以可操作方式連接至至少一調節序列之核苷酸序列的表現載體。調節序列係由技術識別及經選擇以直接表現編碼之多肽。由此，術語調節序列包括啟動子、增強子、及其他表現控制因子。舉例言之，非限制性調節序列係在Goeddel；Gene Expression Technology:Methods in Enzymology
, Academic Press，San Diego，CA(1990)中描述。應瞭解，表現載體之設計可取決於諸如待轉形宿主細胞之選擇及/或期望表現之蛋白質類型之因素。此外，亦應考慮載體之複製數量、控制由載體編碼之任何其他蛋白質(諸如抗生素標記物)之複製數量及表現之能力。
製備本發明抗體之方法可包括，例如，可在容許抗體表現發生之適當條件下培養經編碼抗體之一或數個表現載體(例如，編碼重及輕鏈或其可變區之單一載體、或兩個載體，一者編碼重鏈及一者編碼輕鏈或其可變區)轉染的宿主細胞。可從細胞與含有該抗體之培養基的混合物中分泌及單離該抗體。或者，該抗體可保留在細胞質或膜部分中及細胞收穫、裂解及蛋白質單離。細胞培養物包括宿主細胞、培養基及其他副產物。適宜用於細胞培養之培養基係在此項技術中為吾人所熟知。該抗體可使用在純化蛋白質、抗體、及其抗原結合抗體片段之技術中已知之技術(包括離子交換層析法、凝膠過濾層析法、超微過濾、電泳、及免疫親和性純化)從細胞培養基、宿主細胞、或二者中單離。在某些態樣中，抗體係製備為包括重及輕鏈可變區之抗體之抗原結合片段(其可增加溶解性及促進純化)。
可藉由將選殖基因、或其部分連接至適宜用於在原核細胞、真核細胞(酵母、鳥類、昆蟲或哺乳動物)、或二者中表現之載體來製備重組核酸。用於製備重組多肽之表現媒劑包括質體及其他載體。例如，適宜載體包括以下類型之質體：用於在原核細胞(諸如大腸桿菌)中表現之pBR322-衍生之質體、pEMBL-衍生之質體、pEX-衍生之質體、pBTac-衍生之質體及pUC-衍生之質體。在某些態樣中，哺乳動物表現載體含有促進載體在細菌中增殖之原核序列，及一或數個在真核細胞中表現之真核轉錄單元。該pcDNAI/amp、pcDNAI/neo、pRc/CMV、pSV2gpt、pSV2neo、pSV2-dhfr、pTk2、pRSVneo、pMSG、pSVT7、pko-neo及pHyg衍生之載體係適宜用於轉染真核細胞之哺乳動物表現載體之實例。一些此等載體經獲自細菌質體(諸如pBR322)之序列修飾以促進在原核及真核細胞中複製及耐藥性選擇。或者，病毒諸如牛乳頭瘤病毒(BPV-1)、或艾司坦-巴爾(Epstein-Barr)病毒((pHEBo、pREP-衍生及p205)之衍生物可用於在真核細胞中短暫表現蛋白質。製備質體及轉形宿主生物採用之各種方法係在此項技術中為吾人所熟知。關於原核及真核細胞之其他適宜表現系統，以及一般重組程序，參見Molecular Cloning A Laboratory Manual
，第二版，由Sambrook, Fritsch與Maniatis編輯(Cold Spring Harbor Laboratory Press，1989)第16及17章。在一些實例中，期望藉由使用桿狀病毒表現系統來表現重組多肽。此等桿狀病毒表現系統之實例包括pVL-衍生之載體(諸如pVL1392、pVL1393及pVL941)、pAcUW-衍生之載體(諸如pAcUW1)、及pBlueBac-衍生之載體(諸如含有ß-gal之pBlueBac III)。
用於製備融合基因之技術係為吾人所熟知。基本上，編碼不同多肽/抗體序列之各種核酸片段之接合係依照習知技術進行，採用用於連接、限制酶消解之平末端或交錯末端終點以提供適當終點，按需要填充黏性末端，鹼性磷酸酶處理以避免不期望接合及酶連接。在另一態樣中，可藉由包括自動DNA合成儀之習知技術合成融合基因。或者，可使用在兩連續核酸片段間產生互補懸垂體(其可隨後經退火以產生嵌合基因序列)之錨定引物進行基因片段之PCR擴增(參見，例如，Current Protocols in Molecular Biology
，Ausubel等人編輯，John Wiley & Sons:1992)。
在一些態樣中，表現任何本文描述之核酸的表現載體可用於在宿主細胞中表現抗體。例如，抗體可在細菌細胞諸如大腸桿菌、昆蟲細胞(例如，使用桿狀病毒表現系統)、酵母、或哺乳動物細胞中表現。其他適宜宿主細胞係為熟習此項技術者所熟知。
一旦表現載體藉由習知技術轉移至宿主細胞，該經轉染之細胞隨後藉由習知技術培養以產生抗體。由此，本發明包括含有編碼抗體或其片段之多核苷酸的宿主細胞，以可操作方式連接至異源啟動子。在某些態樣中，重鏈及輕鏈及/或重及輕鏈可變區可在宿主細胞中共同表現(自相同或不同載體)以表現全部抗體。在某些態樣中，抗體之重及輕鏈可自單一啟動子表現。在某些態樣中，抗體之重及輕鏈可自多個啟動子表現。在某些態樣中，該抗體之重及輕鏈可在單一載體上編碼。在某些態樣中，抗體之重及輕鏈可在多個載體上編碼。
可用作表現重組抗體之宿主的哺乳動物細胞系係在此項技術中為吾人所熟知及包括獲自美國菌種保存中心(ATCC)之眾多永生細胞系，包括(但不限於)中國倉鼠卵巢(CHO)細胞、海拉(HeLa)細胞、幼倉鼠腎(BHK)細胞、猴腎臟細胞(COS)、人類肝細胞癌細胞(例如，Hep G2)、人類上皮腎臟293細胞、及數個其他細胞系。不同宿主細胞具有後轉譯處理及修飾蛋白質及基因產物之特徵及特異性機制。可選擇適當細胞系或宿主系統以確保正確修飾及處理表現之抗體或其部分。為此，可使用具有適當處理基因產物之初級轉錄、糖苷化、及磷酸化之細胞機制的真核宿主細胞。此等哺乳動物宿主細胞包括(但不限於)CHO、VERY、BHK、Hela、COS、MDCK、293、3T3、W138、BT483、Hs578T、HTB2、BT2O及T47D、NS0(不內源性地產生任何功能免疫球蛋白鏈之鼠類骨髓癌細胞系)、SP20、CRL7O3O及HsS78Bst細胞。在一態樣中，藉由使人類淋巴細胞永生開發之人類細胞系可用於重組製備單株抗體。在一態樣中，人類細胞系PER.C6.(Crucell，Netherlands)可用於重組製備單株抗體。
可用作表現重組抗體之宿主的額外細胞系包括(但不限於)昆蟲細胞(例如，Sf21/Sf9，Trichoplusia ni
Bti-Tn5b1-4)或酵母細胞(例如，釀酒酵母(S. cerevisiae
)、畢赤酵母屬(Pichia
)、US 7326681；等等)、植物細胞(US20080066200)；及雞細胞(WO2008142124)。
在某些態樣中，本發明抗體係在細胞系中穩定表現。穩定表現可用於長期、高產率製備重組蛋白質、抗體及其抗原結合片段。例如，可產生穩定表現該抗體分子之細胞系。宿主細胞可經包括表現控制因子(例如，啟動子、增強子、轉錄終止區、多腺苷酸化位點等等)及可選擇標記基因之適當工程改造之載體轉形。在引入外源DNA後，可容許細胞在強化型培養基中生長1至2天，及隨後切換到選擇性培養基。在重組質體中之可選擇標記賦予選擇抗性及容許將質體穩定整合至其染色體的細胞生長及形成變異區，其繼而可經選殖及擴展為細胞系。以高產率製備穩定細胞系之方法係在此項技術中為吾人熟知，且試劑一般係市售的。
在某些態樣中，本發明抗體在細胞系中短暫表現。短暫轉染係其中引入細胞之核酸不整合至該細胞之基因組或染色體DNA的過程。其實際上作為細胞中染色體外元件(諸如附加體)維持。附加體之核酸之轉錄過程不受影響及產生由該附加體之核酸編碼之蛋白質。
經穩定或短暫轉染之細胞系係在此項技術中為吾人熟知之細胞培養基及條件下維持，從而產生單株抗體之表現及製備。在某些態樣中，哺乳動物細胞培養基係基於市售培養基調配物，包括，例如，DMEM或漢姆氏F12(Ham's F12)。在其他態樣中，改良該細胞培養基以提供在細胞生長及生物蛋白質表現方面之增長。如本文使用，術語「細胞培養基」、「培養基」、及「培養基調配物」指在多細胞生物或組織外部人工體外環境中維持、生長、增殖、擴展細胞之營養液。細胞培養基可針對特異性細胞培養用途優化，包括例如，經調配以促進細胞生長之細胞生長培養基、或經調配以促進重組蛋白質產生之細胞生產培養基。本文可交換使用術語營養劑、成分、及組分以指構成細胞培養基之部分。
一旦已產生分子，其可藉由任何在純化免疫球蛋白分子或其片段之技術中已知之方法，例如，藉由層析法(例如，離子交換、親和性(特定言之藉由對特異性抗原蛋白質A或蛋白質G之親和性)、及尺寸排阻管柱層析法)、離心、差別溶解度、或藉由任何其他用於純化蛋白質、抗體、及/或抗體片段之標準技術純化。此外，本發明之分子或其片段可融合至本文描述或其他在此項技術中熟知促進純化之異源多肽序列(本文稱為「標籤」諸如組胺酸標籤)。
當使用重組技術時，分子可在細胞內、胞外質空間製備，或直接分泌至培養基。若在細胞內製備該分子，作為第一步驟，例如，藉由離心或超濾移除顆粒碎片(宿主細胞或裂解片段)。Carter等人，Bio/Technology
，10:163-167 (1992)描述一種單離分泌至大腸桿菌胞外質空間之抗體的程序。在分子分泌至培養基之情況下，一般首先使用市售蛋白質濃度過濾器(例如，Amicon或Millipore Pellicon超濾單元)濃縮此等表現系統之上清液。蛋白酶抑制劑諸如PMSF可包含於任何前述步驟以抑制蛋白水解及抗生素可包含在內以防止額外污染物生長。
可單獨或與其他純化步驟組合使用(例如)之氫氧磷灰鹽層析法、疏水相互作用層析法、離子交換層析法、凝膠電泳、透析、及/或親和性層析來純化由細胞製備之組合物。作為親和性配位體之蛋白質A之適合性取決於分子中任何免疫球蛋白Fc域之種類及同型物(若存在)，及將為熟習此項技術者所瞭解。附接親和性配位體之基質最通常係瓊脂糖，但其他基質可用。機械穩定基質諸如受控孔玻璃或聚(苯乙烯二乙烯)苯可達到與瓊脂糖相比容許更快流動速度及更短處理時間。其他蛋白質純化技術諸如在離子交換柱上分餾、乙醇沉澱、反相HPLC、二氧化矽層析、肝素層析、陰離子或陽離子交換樹脂(諸如聚天冬胺酸管柱)SEPHAROSE層析、層析聚焦、SDS-PAGE、及硫酸銨沉澱亦係可用，端視待回收之分子而定。
在任何預備純化步驟後，包括所關注分子及污染物之混合物可接受使用洗脫緩衝液於介於約2.5至4.5之pH之低pH疏水交互層析，及於低鹽濃度(例如，約0至0.25M鹽)下進行。
可使用(例如)上文及/或在實例中列出技術之任一或組合製備及純化抗體。與如何純化抗體無關，為確認本發明抗體之功能結合，可(在純化前及/或後)進行結合分析。例如，可使用雙重ELISA分析。在一些態樣中，將第一抗原(例如，替格瑞洛或其競爭物)塗覆在孔上，及結合至此抗原固定製備其之抗體以用於檢測。 醫藥調配物
在某些態樣中，本發明提供醫藥組合物。此等醫藥組合物可係包括編碼抗體之核酸分子的組合物。此等醫藥組合物亦可係包括抗體、或抗體之組合、及醫藥上可接受之賦形劑之組合物。在某些態樣中，本發明之醫藥組合物係用作藥劑。
在某些態樣中，抗體或抗體之組合(或編碼抗體或抗體之組合之核酸分子)可與醫藥上可接受之載劑、賦形劑或穩定劑調配為醫藥組合物。在某些態樣中，此等醫藥組合物係適宜經由任何一或數個投藥途徑使用在此項技術中熟知之方法投與給人類或非人動物。如熟習此項技術者瞭解，投藥途徑及/或方式可視所需結果變化。術語「醫藥上可接受之載劑」指一或數個不干擾活性成分生物活性之有效性的非毒性物質。此等製劑可通常含有鹽、緩衝劑、防腐劑、相容載劑、及視情況可選之其他治療劑。此等醫藥上可接受之製劑亦可含有適宜投與人類之相容固體或液體填充劑、稀釋劑或封裝物質。可用於本文描述之調配物的其他預期載劑、賦形劑、及/或添加劑包括(例如)調味劑、抗微生物劑、甜味劑、抗氧化劑、抗靜電劑、脂質、蛋白質賦形劑諸如血清白蛋白、凝膠、酪蛋白、鹽形成抗衡離子諸如鈉及等等。適宜用於本文描述之調配物之此等及額外熟知之醫藥載劑、賦形劑及/或添加劑係在此項技術中熟知，例如，如在「Remington: The Science & Practice of Pharmacy」，第21版，Lippincott Williams & Wilkins，(2005)、及「Physician’s Desk Reference」，第60版，Medical Economics，Montvale, N.J.(2005)中列出。可選擇適宜用於期望或需要之投藥模式、溶解性及/或穩定性的醫藥上可接受之載劑。
本文描述之調配物包括以產生w/v適用於期望劑量之濃度的活性劑(例如，抗體或抗體片段諸如Fab或scFv)。在某些態樣中，該活性劑係以約1 mg/ml至約200 mg/ml、約1 mg/ml至約100 mg/ml、約1 mg/ml至約50 mg/ml、或約1 mg/ml至約25 mg/ml之濃度存在於調配物中。在某些態樣中，該活性劑係以約25 mg/ml之濃度存在。在某些態樣中，在調配物中該活性劑濃度可從約0.1至約100重量%變化。在某些態樣中，該活性劑濃度係在0.003至1.0莫耳之範圍內。
在一態樣中，本發明調配物係實質上不具有內毒素及/或相關熱原性物質之無熱原調配物。內毒素包括侷限於微生物內部及僅當該微生物破碎或死亡時釋放之毒素。熱原性物質亦包括源自細菌或其他微生物外膜之引起發熱、熱穩定物質(糖蛋白)。若此等物質兩者投與人類均可引起發熱、低血壓及休克。由於潛在有害效應，甚至少量內毒素必須從靜脈內投與之醫藥藥品溶液中移除。美國食品與藥品管理局(「FDA」)已設置在靜脈內藥物應用一小時期間5內毒素單位(EU)/劑量/千克體重之上限(The United States Pharmacopeial Convention，Pharmacopeial Forum 26 (1):223 (2000))。在某些特定態樣中，在組合物中內毒素及熱原含量係小於10 EU/mg、或小於5 EU/mg、或小於1 EU/mg、或小於0.1 EU/mg、或小於0.01 EU/mg、或小於0.001 EU/mg。
當用於體內投藥時，本發明調配物應滅菌。本發明調配物可藉由各種滅菌方法滅菌，包括無菌過濾、輻射等等。在一態樣中，該調配物經預滅菌之0.22微米過濾器過濾滅菌。可根據習知醫藥實踐(如在「Remington: The Science & Practice of Pharmacy」，第21版，Lippincott Williams & Wilkins，(2005)中所描述)調配用於注射之無菌組合物。
本發明治療組合物可針對特定投藥途徑(諸如口服、經鼻、肺、局部(包括經頰及舌下)、直腸、***及/或非經腸投藥)進行調配。如文中所用，短語「非經腸投藥」及「經非經腸方式投與」指除經腸及局部投藥外之投藥模式，通常藉由注射，且包括(但不限於)經靜脈內、經肌內、經動脈內、經鞘內、經莢膜內、經眼窩內、經心臟內、經真皮內、經腹膜內、經氣管、經皮下、經表皮下、經關節內、莢膜下、蛛網膜下、經脊柱內、經硬膜外及經胸骨內注射及輸注。適宜局部或經皮投藥之本發明調配物包括粉劑、噴霧劑、軟膏、糊劑、乳膏、乳液、凝膠、溶液、貼片及吸入劑。該抗體可在滅菌條件下與醫藥上可接受之載劑、及任何可需要之防腐劑、緩衝劑、或推進劑混合(美國專利第7,378,110號；第7,258,873號；第7,135,180號；美國公開案第2004-0042972號；及第2004-0042971號)。
該調配物可方便地以單位劑型存在及藉由藥學技術中任何已知的方法製備。在本發明之醫藥組合物中活性成分之實際劑量水平可變化以獲得有效達成針對特定患者、組合物及投藥模式之期望治療反應，而對患者不具有毒性的活性成分量(例如，「治療有效量」)。經選擇劑量水平將取決於多種藥物動力學因素，其包括採用之特定組合物之活性、投藥途徑、投藥時間、採用之特定化合物之排洩速度、治療持續期、其他藥物、與採用之特定組合物組合使用之化合物及/或材料、待治療患者之年齡、性別、體重、狀態、一般健康及先前藥物史及在藥物技術中熟知之類似因素。適宜劑量可從約0.0001至約100 mg/kg體重或更高變化，例如約0.1、1、10、或50 mg/kg體重，約1至約10 mg/kg體重係適宜。
應注意，本發明相似地設計，亦可製備適用於診斷及研究使用之調配物。可基於特定應用及預期用途選擇在此等調配物中之活性劑濃度，以及存在或缺乏賦形劑及/或熱原。 用途
本文揭示之抗體適用於包括組合治療之治療方法，其用於中和血小板活化、凝聚及脫粒抑制劑、血小板解聚促進劑、及抗血栓形成劑之活性。由此，發現本文描述之抗體可用於數個有關投與替格瑞洛之應用(包括伴隨方法)及係適宜用於中和替格瑞洛及/或替格瑞洛之一或數個代謝物之效應。在此等方法中，該抗體可視情況可逆地降低、中和、消除、或以其他方式抑制替格瑞洛之活性及治療或預防與替格瑞洛投藥相關聯及/或產生自包括替格瑞洛之治療的任何數量之效應、疾病及/或症狀。
該抗體可投與待治療或需要治療或預防替格瑞洛(BRILINTA)可治療及/或指明之適應症的患者，該等適應症包括(例如)不穩定心絞痛、動脈粥樣硬化之原發性動脈血栓形成併發症，諸如血栓形成或栓塞性中風、短暫性腦缺血發作、周邊血管疾病、具有或不具有血栓形成之心肌梗塞、由於動脈粥樣硬化疾病之干預諸如血管成形術(包括冠狀動脈成形術(PTCA)、動脈內膜切除術、支架放置、冠狀動脈及其他血管移植手術)所致的動脈併發症、外科手術或機械損傷(諸如在事故或手術創傷之後的組織補救、包括皮膚及肌瓣之整形手術)的血栓形成併發症、在瀰漫性血栓形成/血小板消耗組分之病狀諸如廣泛性血管內凝固、血栓形成性血小板減少症性紫癜、溶血症候群、敗血病之血栓形成併發症、成人呼吸窘迫症候群、抗磷脂症候群、肝素引發之血小板減少症及預驚厥/驚厥、或靜脈血栓形成諸如下肢深靜脈血栓、靜脈閉塞疾病、血液病狀諸如骨髓增生性疾病，包括血小板增多症、鐮狀细胞疾病；或預防機械引發之體內血小板活化，諸如心肺旁路及體外膜氧合(預防微小血栓栓塞症)、機械引發之體外血小板活化，諸如用於保存血液產品，例如，血小板濃縮物、或分流閉塞(諸如在腎透析及血漿除去法)，血管損傷/炎症諸如脈管炎、動脈炎、血管球性腎炎、炎性腸病及器官移植排斥反應，症狀(諸如偏頭痛)繼發之血栓。
在一些實施例中，本文揭示之抗體可投與給將要接受或已接受包括替格瑞洛之治療，及需要治療或將要需要治療與以下相關聯之出血或潛在出血之患者：冠狀動脈繞道移植手術(CABG)、胸心手術、縱膈再探、術後中風、機械換氣法、在加護病房中長期停留、非心臟外科手術(例如，神經或眼科手術、腎柱外科手術、顱內手術、眼眶手術、矯形外科手術、腎切除術、部分結腸切除術等等)。因此，本文提供之方法可涵蓋與替格瑞洛共同治療(同時)、或在投與替格瑞洛後一段時間(例如，數分鐘、數小時、或數天)內之抗體投與。例如，在一些實施例中，該方法可包括在投與替格瑞洛10至120分鐘內將該抗體投與給已經投與替格瑞洛之患者。在一些實施例中，該方法可包括在投與替格瑞洛1至48小時內將該抗體投與給已經投與替格瑞洛之患者。在一些實施例中，在不容許替格瑞洛及/或其代謝物從受試者新陳代謝及消除之一定時間內將該抗體投與給已經投與替格瑞洛之受試者。
在一些實施例中，本發明提供一種抑制替格瑞洛或其活性代謝物對患者之(P2Y12
)受體之效應之方法。
在一些實施例中，本發明提供一種抑制替格瑞洛或其活性代謝物結合至患者之P2Y12
受體之方法。
在一些實施例中，本發明提供一種在已經投與替格瑞洛之患者中活化ADP誘發之血小板凝聚之方法。
本發明之抗體(諸如在實例中例示之彼等)亦可用於診斷目的。例如，可在受試者之組織或細胞中檢測一或數個靶試劑(替格瑞洛或其代謝物)以測定或篩選受試者中替格瑞洛之循環量。診斷套組可包括一或數個抗體、及用於指示抗體與替格瑞洛或其代謝物(若任一者存在)反應之檢測系統。
由此，本發明設計抗體之數個用途，包括治療、診斷及研究用途。診斷及研究用途可係體內或體外。 套組
本發明之另一態樣係一種套組。在一態樣中，套組包括上文描述之核酸、抗體、表現載體、或宿主細胞的組合物或醫藥組合物中之任一者，及引導適當使用或投藥之說明書或標識。視情況，套組亦包括一或數個容器及/或注射器或其他裝置以促進遞送或使用。本發明設計用於進行研究分析、診斷分析及/或用於投與治療有效量之組分之全部或任何子集係封裝於套組中。相似地，該套組可包括用於藉由(例如)在適宜條件下培養表現編碼本發明抗體之核酸的宿主細胞製備抗體之說明。舉額外實例言之，用於治療性投與本發明抗體之套組可包括含有該抗體之醫藥調配物的溶液、或該抗體之凍乾製劑、及將該組合物按投與給有需要之患者及/或用於復水該凍乾產品之說明。在某些實施例中，該套組進一步包括在適宜投與給受試者之調配物中的替格瑞洛(例如，BRILINTA™、BRILIQUE™)。在此等實施例中，該套組進一步包括用於投與抗體及替格瑞洛調配物二者給有經抗體、經替格瑞洛、或經抗體及替格瑞洛二者治療需要之患者的說明。
本發明亦涵蓋最終包裝及標識之醫藥產品。此製品包括在適當容器或容納物諸如玻璃瓶或其他氣密密封容器中之適當單位劑型。就適宜於非經腸投與該活性成分(例如，上文描述之抗體及/或替格瑞洛調配物)之劑型而言，係滅菌及適宜作為無顆粒溶液投藥。在某些態樣中，該調配物適宜用於可注射投藥途徑。在一些實施例中，該投藥係皮下。在一些實施例中，該投藥係靜脈內投藥。由此，設計包括注射或輸注至人類或動物的投藥途徑。
在一特殊態樣中，本發明調配物係在單一劑量瓶中調配為無菌液體。例示性容器包括(但不限於)小瓶、瓶、預填充注射器、IV袋、氣泡包裝(包括一或數個丸劑)。視情況與此(等)容器相關聯的係以由管理藥品或生物產品之製造、使用或銷售的政府機構規定之形式的注意事項，其注意事項反映由製造、使用或銷售用於人類診斷及/或投藥之機構批准。
如同任何醫藥產品，設計包裝材料及容器以在存儲及運輸期間保護該產品之穩定性。此外，本發明產品包括使用說明或建議醫生、技術者人員或患者如何適當預防或治療討論之疾病或病症的其他資訊材料。換言之，製品包括表明或建議包括(但不限於)實際劑量、監測程序等等之投藥方案，及其他監測資訊的說明書構件。
用於診斷分析之套組可包括含有本發明抗體的溶液或抗體之凍乾製劑，其中該抗體特異性結合至替格瑞洛及/或其代謝物，以及用於檢測此抗體之試劑。該抗體可根據在此項技術中熟知及本文描述之方法，包括(但不限於)標識，諸如小分子熒光標籤、蛋白質諸如生物素、GFP或其他熒光蛋白質、或抗原決定基序列諸如his或myc進行標識。相似地，用於檢測抗體之一級抗體可包括於該套組內。一級抗體可指向在抗體上之序列或標識抗體所用之標識、標籤、或抗原決定基。一級抗體可繼而經標識以用於檢測或若需要進一步放大信號，則可藉由二級抗體(其可亦包括於該套組內)檢測一級抗體。
亦設計用於研究用途之套組。此等套組可(例如)類似於預期用於診斷或治療用途之套組但進一步包括指明將該套組及其用途僅限於研究目的之標識。實例 縮寫清單 縮寫 解釋
ACN 乙腈
Br 寬
BSA 牛血清白蛋白
CV 管柱容積
d 雙重
dd 雙雙重
DCM 二氯甲烷
DMF N,N-二甲基甲醯胺
DMSO 二甲亞碸
DPBS 杜貝卡氏磷酸鹽緩衝鹽水
EDC 1-乙基-3-(3-二甲基胺基丙基)碳化二亞胺；
EtOAc 乙酸乙酯
FA 甲酸
HOAc 乙酸
HPLC 高效液相層析
HRMS 高解析度質譜分析
HTS 高通量篩選
HTRF®
均相時差式熒光
HYFLO®
助濾劑，助熔煅燒，經碳酸鈉處理
Hz 赫茲
J 偶合常數
LC 液相層析
m 多重
MS 質譜分析
NMR 核磁共振
OAc 乙酸鹽
Pd/C 炭上鈀
pM 皮莫耳
PK/PD 藥代動力學/藥效動力學
KF 氟化鉀
q 四重
r.t. 室溫
s 單重
sat 飽和
scFv 單鏈可變片段
t 三重
TFA 三氟乙酸
TEA 三乙胺
TBME 第三丁基甲基醚
THF 四氫呋喃
TIM 替格瑞洛非活性代謝物
TLC 薄層層析
TR-FRET 時差式熒光共振能量轉移實例 1 ：半抗原之製備及特性分析
此實例描述合成、優化、單離、及特性分析若干用於產生如本文描述之例示性抗體之半抗原的方法。該等半抗原包括替格瑞洛、替格瑞洛代謝物(TAM及TIM)、生物素化之替格瑞洛、及生物素化之腺苷(參見，例如，呈未生物素化之形式的化學半抗原結構之圖2)。如在國際專利公開案WO 2000/034283(Guile等人，2000)中所描述合成替格瑞洛及如在國際專利公開案WO 1999/005143(Guile等人，1999)中所描述合成TAM，各者其全文以引用的方式併入本文。
使用Biotage矽膠40S、40M、12i或Merck矽膠60(0.063至0.200 mm)進行直相層析。使用標準玻璃或塑料管柱或在Biotage Horizon系統上進行急驟層析。使用溶劑作為內標物給出以ppm計之化學位移。僅當在NMR中檢測時記錄在雜原子上之質子諸如NH
及OH
質子及可由此忽視。實例 1.1 ：生物素化之替格瑞洛
N-(2-(((1S,2S,3S,4R)-4-(7-(((1R,2S)-2-(3,4-二氟苯基)環丙基)胺基)-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-3-基)-2,3-二羥基環戊基)氧基)乙基)-6-(6-(5-((3aS,4S,6aR)-2-側氧基六氫-1H-噻吩并[3,4-d]咪唑l-4-基)戊醯胺基)己醯胺基)己醯胺(1.1
)
(i)製備甲磺酸2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-二氟苯基)環丙基)胺基-5-(丙硫基-3H-[1,2,3]***并[4,5-d]嘧啶-3-基)-2,2-二甲基四氫-3aH-環戊[d][1,3]二氧雜環戊烯-4-基)氧基)乙酯(1.a
)
於0℃下，將甲磺醯氯(0.086 mL，1.10 mmol)逐滴添加至2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-二氟苯基)環丙基)胺基)-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-3-基)-2,2-二甲基四氫-3aH-環戊[d][1,3]二氧雜環戊烯-4-基)氧基)乙醇(參見Springthorpe, B.等人Bioorg.Med. Chem. Lett.,，2007，17，6013-6018)(0.563 g，1.0 mmol)與TEA(0.209 mL，1.50 mmol)在DCM(5 mL)中之溶液。歷時3 h將該混合物從0℃攪拌至約5℃。使用DCM(30 mL)稀釋該反應混合物及使用水(5 mL)洗滌。藉由經過相分離器乾燥該混合物。蒸發溶劑及自甲苯共蒸發獲得呈黃色稠油之標題化合物(1.a
)(714 mg，111%)，其用作粗製物而不經進一步純化。1
H NMR (400 MHz, CDCl3
) δ 1.02 (dd, 3H), 1.3 – 1.47 (m, 5H), 1.55 (s, 3H), 1.72 (d, 2H), 2.20 (d, 1H), 2.6 – 2.71 (m, 2H), 2.97 (s, 3H), 3 – 3.19 (m, 3H), 3.57 – 3.68 (m, 1H), 3.69 – 3.79 (m, 1H), 4.02 (td, 1H), 4.13 – 4.24 (m, 2H), 4.78 (dd, 1H), 5.13 (td, 1H), 5.57 (s, 1H), 6.50 (s, 1H), 7.03 (s, 1H), 7.07 – 7.16 (m, 2H)。19
F NMR (376 MHz, CDCl3
) δ -141.37 (J = 21.3), -138.10 (J = 21.3)。
(ii)製備3-((3aS,4R,6S,6aR)-6-(2-疊氮基乙氧基)-2,2-二甲基四氫-3aH-環戊[d][1,3]二氧雜環戊烯-4-基)-N-((1R,2S)-2-(3,4-二氟苯基)環丙基)-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-7-胺(1.b
)
將甲磺酸2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-二氟苯基)環丙基)胺基)-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-3-基)-2,2-二甲基四氫-3aH-環戊[d][1,3]二氧雜環戊烯-4-基)氧基)乙酯(1.a)(0.641 g，1 mmol)及疊氮化鈉(0.070 mL，2.00 mmol)在DMF (7 mL)中之混合物在氮氣氛圍下加熱至60℃持續15.5 h。形成白色沉澱。添加水(20 mL)及使用TBME(100+40 mL)萃取該產物兩次。經Na2
SO4
乾燥有機相。過濾有機相及於減壓下移除該溶劑。藉由急驟層析法在2 x 8 cm二氧化矽管柱上使用庚烷/EtOAc 1/1作為溶離劑純化該殘留物(TLC，其使用庚烷/EtOAc 1/1(Rf產物=0.5))。收集相關溶離份及蒸發該溶劑獲得呈透明稠油之標題化合物(1,b
)(514 mg，87%)。1
H NMR (400 MHz, CDCl3
) δ 1.00 (s, 3H), 1.33 – 1.42 (m, 5H), 1.59 (s, 3H), 1.73 (d, 2H), 2.17 (s, 1H), 2.68 (t, 2H), 2.96 – 3.17 (m, 3H), 3.19 – 3.33 (m, 2H), 3.52 – 3.63 (m, 1H), 3.72 (ddd, 1H), 4.03 (td, 1H), 4.79 (dd, 1H), 5.13 (td, 1H), 5.54 (dd, 1H), 6.43 (s, 1H), 6.96 – 7.23 (m, 3H)。
(iii)製備中間產物3-((3aS,4R,6S,6aR)-6-(2-胺基乙氧基)-2,2-二甲基四氫-3aH-環戊[d][1,3]二氧雜環戊烯-4-基)-N-((1R,2S)-2-(3,4-二氟苯基)環丙基)-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-7-胺(1.c
)
將在EtOH(99.5 %)(2 mL)中之3-((3aS,4R,6S,6aR)-6-(2-疊氮基乙氧基)-2,2-二甲基四氫-3aH-環戊[d][1,3]二氧雜環戊烯-4-基)-N-((1R,2S)-2-(3,4-二氟苯基)環丙基)-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-7-胺(1.b
)(62.0 mg，0.11 mmol)添加至Pd/C(5% Pd，50重量% Pd/C，22.46 mg，5.28 µmol)及於大氣壓力下氫化該混合物2 h。經由HYFLO®過濾該反應混合物及進一步使用EtOH(99.5%)沖洗塞柱。在減壓下移除該溶劑，將該殘留物再次溶於DCM(2 x 2 mL)及在減壓下移除該溶劑。藉由急驟層析在2 x 2 cm二氧化矽管柱上使用DCM/NH3
(飽和)在MeOH中之95/5作為溶離劑純化該殘留物。收集相關溶離份獲得標題化合物(1.c
)(41 mg，69%)。1
H NMR (400 MHz, CDCl3
) δ 0.98 (m, 3H), 1.28 – 1.46 (m, 7H), 1.54 (s, 3H), 1.62 – 1.81 (m, 2H), 2.15 (s, 1H), 2.48 – 2.81 (m, 4H), 3.07 (tt, 3H), 3.34 – 3.47 (m, 1H), 3.53 (ddd, 1H), 3.99 (td, 1H), 4.79 (dd, 1H), 5.12 (td, 1H), 5.52 (dd, 1H), 7.02 (s, 1H), 7.09 (dt, 2H), 7.23 (s, 1H)。19
F NMR (376 MHz, CDCl3
) δ -141.43 (J
= 21.3), -138.13 (J
= 21.3)。
(iv)製備中間產物(1S,2S,3S,5R)-3-(2-胺基乙氧基)-5-(7-(((1R,2S)-2-(3,4-二氟苯基)環丙基)胺基)-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-3-基)環戊烷-1,2-二醇(1.d
)
將預冷卻之冰/水浴溫的TFA(8 mL，103.84 mmol)與水(0.88 mL，48.85 mmol)之混合物添加至預冷卻之具有3-((3aS,4R,6S,6aR)-6-(2-胺基乙氧基)-2,2-二甲基四氫-3aH-環戊[d][1,3]二氧雜環戊烯-4-基)-N-((1R,2S)-2-(3,4-二氟苯基)環丙基)-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-7-胺(1.c
)(340 mg，0.61 mmol)之燒瓶中。於0至5℃下攪拌該反應混合物1 h。在減壓下移除該溶劑及將殘留物溶於DCM(100 mL)及使用NaHCO3
(飽和10 mL)洗滌。將鹽水(5 mL)添加至水相及使用EtOAc(30 mL)萃取此。經Na2
SO4
乾燥合併之有機相。過濾，接著蒸發該溶劑獲得米白色固體粗產物。藉由製備型HPLC在XBridge C18管柱(10 μm 250 x 50 ID mm)上使用35至75%梯度之ACN在H2
O/ACN/NH3
95/5/0.2中之緩衝液以100 mL/min之流速純化該化合物20分鐘。藉由UV於298 nm檢測該等化合物。在減壓下將峰溶離份蒸發至乾燥。殘留物溶於DCM及經相分離器過濾。在減壓下移除溶劑，獲得標題化合物(1.d
)(213 mg，67.5%)。LC-MS m/z 522.3(M+H)+
。
(v)製備化合物N-(2-(((1S,2S,3S,4R)-4-(7-(((1R,2S)-2-(3,4-二氟苯基)環丙基)胺基)-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-3-基)-2,3-二羥基環戊基)氧基)乙基)-6-(6-(5-((3aS,4S,6aR)-2-側氧基六氫-1H-噻吩并[3,4-d]咪唑-4-基)戊醯胺基)己醯胺基)己醯胺。(1.1
)
將6-(6-(5-((3aS,4S,6aR)-2-側氧基六氫-1H-噻吩并[3,4-d]咪唑-4-基)戊醯胺基)己醯胺基)己酸2,5-二側氧基吡咯啶-1-基酯(21.77 mg，0.04 mmol)添加至(1S,2S,3S,5R)-3-(2-胺基乙氧基)-5-(7-(((1R,2S)-2-(3,4-二氟苯基)環丙基)胺基)-5-(丙硫基)-3H-[1,2,3]***并[4,5-d]嘧啶-3-基)環戊烷-1,2-二醇(20 mg，0.04 mmol)在無水DMF(1.0 mL)中之溶液及將該混合物置於氮氣氛圍及於室溫下攪拌6 h。於40℃在減壓下移除該溶劑。藉由製備型HPLC在Kromasil C18管柱(10 μm 250 x 20 ID mm)上使用20至60%梯度之ACN在H2
O/ACN/FA 95/5/0.2中之緩衝液以19 mL/min之流速純化該化合物20分鐘。藉由UV於298 nm檢測該化合物。收集、濃縮、及冷凍乾燥峰溶離份以獲得標題化合物(1.1
)(21.4 mg，57.3%)。1
H NMR(600 MHz，DMSO)：存在兩種旋轉異構體(比例5:1)，獲自主要旋轉異構體之信號於δ 0.81 (t, 3H), 1.15 – 1.64 (m, 20H), 2.03 (ddd, 7H), 2.12 (ddd, 1H), 2.57 (d, 1H), 2.59 – 2.67 (m, 1H), 2.77 – 2.89 (m, 2H), 2.93 (dd, 1H), 2.96 – 3.01 (m, 4H), 3.05 – 3.12 (m, 1H), 3.15 (td, 1H), 3.18 – 3.25 (m, 2H), 3.39 – 3.46 (m, 1H), 3.48 (tt, 1H), 3.7 – 3.76 (m, 1H), 3.92 (s, 1H), 4.08 – 4.14 (m, 1H), 4.30 (dd, 1H), 4.54 (dd, 1H), 4.95 (q, 1H), 5.06 (s, 1H), 5.13 (d, 1H), 6.35 (s, 1H), 6.42 (s, 1H), 7.07 (d, 1H), 7.31 (ddt, 2H), 7.71 (dt, 2H), 7.82 (t, 1H), 9.36 (d, 1H)。選擇獲自較少旋轉異構體之信號於δ 0.98 (CH3
), 8.95 (ArNH
)。
[C45
H65
F2
N11
O7
S2
]+
之HRMS計算值：974.4556；實測值：974.4585 (M+H)+ 實例 1.2 ：生物素化之腺苷
N-(((2R,3S,4R,5R)-5-(6-胺基-9H-嘌呤-9-基)-3,4-二羥基四氫呋喃-2-基)甲基)-6-(6-(5-((3aS,4S,6aR)-2-側氧基六氫-1H-噻吩并[3,4-d]咪唑-4-基)戊醯胺基)己醯胺基)己醯胺(1.2
)
(i)製備N-(((3aR,4R,6R,6aR)-6-(6-胺基-9H-嘌呤-9-基)-2,2-二甲基四氫呋喃[3,4-d][1,3]二氧雜環戊烯-4-基)甲基)-6-(6-(5-((3aS,4S,6aR)-2-側氧基六氫-1H-噻吩并[3,4-d]咪唑-4-基)戊醯胺基)己醯胺基)己醯胺(1.e
)
於室溫下，將DMF(2 mL)添加至6-(6-(5-((3aS,4S,6aR)-2-側氧基六氫-1H-噻吩并[3,4-d]咪唑-4-基)戊醯胺基)己醯胺基)己酸2,5-二側氧基吡咯啶-1-基酯(55.6 mg，0.10 mmol)及9-((3aR,4R,6R,6aR)-6-(胺基甲基)-2,2-二甲基四氫呋喃并[3,4-d][1,3]二氧雜環戊烯-4-基)-9H-嘌呤-6-胺(30 mg，0.10 mmol)(參見Austin, D.J.及Liu, F.，Tetrahedr. Lett.，2001
，3153-3154)及將該反應混合物置於氮氣氛圍下及攪拌1 h又45 min以獲得(1.e
)。隨後於減壓下移除該溶劑。用作粗產物而不經進一步純化。LC-MS m/z 759 (M+H)+
，757(M-H)-
。
(ii)製備最終化合物N-(((2R,3S,4R,5R)-5-(6-胺基-9H-嘌呤-9-基-3,4-二羥基四氫呋喃-2-基)甲基)-6-(6-(5-((3aS,4S,6aR)-2-側氧基六氫-1H-噻吩并[3,4-d]咪唑-4-基)戊醯胺基)己醯胺基)己醯胺(1.2
)
將TFA(1.8 ml，23.36 mmol)與水(0.2 mL，11.10 mmol)之混合物添加至粗製N-(((3aR,4R,6R,6aR)-6-(6-胺基-9H-嘌呤-9-基)-2,2-二甲基四氫呋喃[3,4-d][1,3]二氧雜環戊烯-4-基)甲基)-6-(6-(5-((3aS,4S,6aR)-2-側氧基六氫-1H-噻吩并[3,4-d]咪唑-4-基)戊醯胺基)己醯胺基)己醯胺(1.e
)(76 mg，0.1 mmol)及於0℃攪拌該反應混合物1小時25分鐘。在減壓下移除該溶劑及殘留物溶於DMSO。藉由製備型HPLC在XBridge C18管柱(10 μm 250 x 19 ID mm)上使用5至45%梯度之CAN在H2
O/ACN/NH3
95/5/0.2中之緩衝液以19 mL/min之流速純化該化合物20分鐘。藉由UV於259 nm檢測該化合物。濃縮及冷凍乾燥峰溶離份以獲得呈白色酥鬆固體之標題化合物(1.2
)(50 mg，69.6%)。1
H NMR (600 MHz, DMSO, 40°C) δ 1.17 – 1.26 (m, 4H), 1.27 – 1.4 (m, 6H), 1.43 – 1.54 (m, 7H), 1.62 (ddt, 1H), 1.99 – 2.06 (m, 4H), 2.12 (t, 2H), 2.58 (d, 1H), 2.82 (dt, 1H), 2.96 – 3.05 (m, 4H), 3.05 – 3.14 (m, 1H), 3.36 (dt, 1H), 3.44 (dt, 1H), 3.96 (dd, 1H), 4.04 (dd, 1H), 4.11 – 4.15 (m, 1H), 4.29 – 4.33 (m, 1H), 4.67 (dd, 1H), 5.16 (d, 1H), 5.38 (d, 1H), 5.84 (d, 1H), 6.29 (s, 1H), 6.33 (d, 1H), 7.25 (s, 2H), 7.64 (dt, 2H), 8.11 (t, 1H), 8.16 (s, 1H), 8.31 (s, 1H)。針對[C32
H50
N10
O7
S]+
之HRMS計算值：719.3657；實測值：719.3667 (M+H)+ 實例 2 ：單離及鑑定抗替格瑞洛 /TAM 抗體
此實例闡明可用於製備針對替格瑞洛及其代謝物、結構相似於ATP及含有類腺苷核心之化合物之抗體的策略及技術(Springthorpe等人，2007 Bioorg Med Chem Lett. 17:6013-6018)。替格瑞洛、替格瑞洛活性代謝物(TAM)及替格瑞洛非活性代謝物(TIM)之化學結構在圖 2
中顯示。如上文討論，本文揭示及產生之抗體可結合并中和替格瑞洛及TAM及可結合至TIM，但不結合或顯著抑制其他結構相關化合物諸如腺苷。當對TIM之結合活性係本文揭示抗體之可選特徵時，由於TIM通常代表替格瑞洛代謝物之小或不重要部分，所以預期顯示對TIM結合活性之抗體不會影響所需該抗體/解毒劑之劑量。
靶向常用抗原決定基(即替格瑞洛及TAM之獨特R基團(二氟苯基-環丙基及硫丙基取代基))以賦予針對彼等化合物之抗體結合特異性及選擇性。所關注抗原決定基在圖 2
中由虛線圈出。在實例1中描述之半抗原用於將抗體抗原決定基引導朝向二氟苯基-環丙基及硫丙基取代基。如在實例1中描述，用於生物素化之半抗原(生物素化之替格瑞洛及生物素化之腺苷)之連接子位於二元醇基上。此策略容許製備對未修飾之二-氟苯基-環丙基及硫丙基、生物素化之替格瑞洛/TAM具有結合特異性之抗體，及亦能夠用於篩選及取消選擇結合腺苷之抗體庫。
使用已知技術，人類scFv噬菌體呈現庫可用於產生scFv抗體，及特異性scFv在一系列重複選擇生物素化替格瑞洛而取消選擇生物素化腺苷之循環中從庫中單離，基本上如在Lloyd等人，2009 PEDS 22:159-168(以引用的方式併入本文中)中所描述。選擇從循環2及循環3選擇輸出之數個個別純系，scFv在細菌周質中表現及在三個平行生物化學分析中篩選特異性。該分析篩選i)結合至生物素化之替格瑞洛(分析1)，ii)結合至生物素化之腺苷(分析2)及iii)在50倍過量未經修飾替格瑞洛存在下結合至生物素化之替格瑞洛(分析3)以確認對替格瑞洛而非生物素化連接子之特異性。
使用相同一般技術及策略進行分析1、2及3。簡言之，結合至生物素化之替格瑞洛或生物素化之腺苷之粗製周質scFv樣品之HTS係使用HTRF®
分析技術進行。HTRF®
(均相時差式熒光)係基於TR-FRET(時差式熒光共振能量轉移)原理。簡言之，TR-FRET採用從供體熒光團(在此情況下銪穴狀化合物)至受體熒光團(在此情況下XL665
)之能量轉移。條件係供體及受體熒光團係在足夠接近(約＜10 nm)，銪穴狀化合物供體之激發(337 nm)導致能量轉移至XL665
受體，其繼而發射於665 nm下之熒光信號。此技術可用於藉由將供體及受體熒光團(直接或間接)附接至在特定交互作用下之各結合搭檔來敏感地測量生物分子交互作用。scFv結合至生物素化替格瑞洛之HTS形式(分析1)係在下文表示及依賴在生物素化替格瑞洛及his標記之周質scFv兩者中化學標籤之存在：
銪穴狀化合物鏈黴抗生物素蛋白：生物素化之替格瑞洛：scFv-His：抗-His-XL665
在包括DPBS pH 7.4(Gibco 14190-086)、KF(VWR 103444T)(0.4M)及Tween 20(Sigma P9416)(0.05%)之緩衝液中以10 μl之分析體積使用黑色淺孔384孔分析板(Corning/Costar 3676)進行該分析。藉由添加5 μl生物素化之替格瑞洛(60 nM以獲得30 nM最終濃度)、2 μl之周質scFv樣品(20%最終濃度)及3 μl含有銪穴狀化合物標記之鏈黴抗生物素蛋白(CisBio 610SAKLB)(4.2 nM以獲得1.26 nM最終濃度)及XL665
標記之抗-His抗體(CisBio 61HISXLB)( 40 nM以獲得12 nM之最終濃度)之溶液建立該分析。建立除添加2 μl分析緩衝液代替周質scFv處外含有全部上文分析組分之陰性結合對照孔。該分析板於室溫下培養4小時，然後使用其中樣品於337 nm激發及時差熒光發射於620 nm及665 nm測量之標準HTRF讀取方案在Envision板讀取器上讀取。
原始665 nm及620 nm計數首先轉化為665 nm/620 nm比值及隨後結果表示為ΔF(%)值。根據以下等式計算ΔF：
ΔF(%)={((樣品665/620比)-(陰性665/620比))/(陰性665/620比例)}x100
(採用獲自陰性結合對照孔之陰性比例)。在此分析中提供大於100%之ΔF值的scFv定義為命中物(hit)。
如上文描述之相同方案用於進行粗製周質scFv樣品對結合至生物素化腺苷之HTS(最終分析濃度30 nM)。分析2之形式可如下列出描繪：
銪穴狀化合物鏈黴抗生物素蛋白：生物素化之腺苷：scFv-His：抗-His-XL665
分析3採用如上文描述之相同方案以進行鑑定在過量游離未經修飾替格瑞洛存在下顯示對生物素化替格瑞洛減低結合之粗製周質scFv樣品的HTS。此方案由分析1改良，在於在50倍莫耳過量之游離未經修飾替格瑞洛(1500 nM)存在下進行分析3。
命中物定義為結合至生物素化之替格瑞洛(在分析1中ΔF＞100%)，而不結合至生物素化之腺苷(在分析2中ΔF＜25%)及在過量游離未經修飾之替格瑞洛存在下＞50%減低之結合至生物素化之替格瑞洛。分析1及3數據之相關性之一實例在圖 3
中顯示。在過量游離未經修飾之替格瑞洛存在下，數個scFv顯示受限抑制，表明其與替格瑞洛及連接子之某些組分具有結合相互作用。在過量游離之未經修飾替格瑞洛存在下，鑑定抑制(＞50%)對生物素化替格瑞洛之結合的scFv之子集。基於在分析3與分析1中觀察到之結合抑制%(50至80%、80至90%、＞90%)分類scFv，其中提供＞90%抑制之scFv(包括TICA0072)優先用於進一步特性分析。將序列獨特scFv 命中物轉化為Fab及使用標準技術在CHO細胞中表現。Fab 表現及純化
獨立之HC及LC表現質體用於暫時轉染，基於由Persic等人1997描述之表現載體。修飾該載體以含有EBV複製起點(OriP
)。該Fab(HC)載體僅含有恆定區1(CH1)及鉸鏈區，CH2及CH3被移除。根據製造商建議將HC及LC DNA添加至150 mM NaCl及25-kDa直鏈PEI(Polysciences Europe，Germany 23966)。隨後將DNA-PEI複合物添加至適合懸浮培養之由CHOK1細胞系(ECACC編號：85051005)衍生之中國倉鼠卵巢野生型(CHO wt)細胞(Daramola O等人2014)。7天後，藉由離心收穫該細胞及過濾上清液。該含有Fab蛋白質之細胞培養上清液直接裝載至於5 ml/min之流速下使用Äkta純化儀(GE Healthcare)之填充有5 ml CaptureSelect IgG-CH1(Life Technologies, Carlsbad，USA)之層析管柱。根據樹脂製造商之說明，平衡該管柱及使用pH 7.2之經磷酸鹽緩衝之鹽水(PBS)洗滌及使用20 mM檸檬酸鈉、150 mM氯化鈉，pH 3.5(CaptureSelect IgG-CH1)洗脫。在分析前將洗脫之Fab調整至pH 5.5及過濾(0.22 μm Steriflip，Millipore EMD，Bethdesa, USA)。藉由於280 nm之吸光度使用DU520 UV/vis光譜儀(Beckman Coulter，Brea，USA)測定蛋白質濃度。使用TSK凝膠G3000SWxl管柱(Tosoh Bioscience，Tokyo，Japan)及於1.0 ml/min下運行之1100 HPLC系統(Agilent Technologies，Santa Clara，USA)測定樣品純度。實例 3 ：抗替格瑞洛 /TAM 之 Fab
詢問含有市售藥物之結構資料庫(「DrugsDB」，Oprea T.I等人，2011 Mol. Inform. 30(2-3)，100-111，以引用的方式併入本文中)以確定具有替格瑞洛之某些結構相似性之分子。一旦確定，此等結構相似分子用於測試除腺苷及其磷酸化形式(例如，ADP及ATP)外Fab之結合特異性。詢問該資料庫具有與替格瑞洛2D指紋相似性、3D形狀、及靜電相似性之分子，基於替格瑞洛X-射線及NMR結構。從此電腦分析中，選擇包括六種潛在協同藥物之一組12種化合物。此等化合物之結構在圖 4
中顯示。
於競爭結合分析形式中研究特異性，其中測試各個測試化合物競爭性抑制生物素化替格瑞洛與相關Fab之交互作用之能力或其他方面。HTRF®
競爭分析形式如下文列出使用，其中目的係藉由一組測試化合物測量結合至各個His-Fab之生物素化替格瑞洛之競爭：
銪穴狀化合物抗His抗體：測試His-Fab：生物素化之替格瑞洛：XL665
標識之鏈黴抗生物素蛋白。
此基本分析形式用於評估獲自先導分離及先導優化期結束時之先導Fab的選擇性曲線及出於此等研究之目的使用His-Fab表現載體產生Fab。
在黑色淺孔384孔分析板(Corning/Costar 3676)中以20 μl分析體積在包括DPBS pH 7.4(Gibco 14190-086)、KF(VWR 103444T)(0.4M)及BSA(PAA K05-013)(0.1%)的緩衝液中進行該分析。涉及添加5 μl生物素化之替格瑞洛、5 μl各個測試選擇性化合物之滴定、5 μl相關His-Fab及5 μl含有銪穴狀化合物標識之抗-His抗體(CisBio 61HISKLB)(5.33 nM以獲得1.33 nM之最終濃度)及XL665
標識之鏈黴抗生物素蛋白(CisBio 611SAXLB)(40 nM以獲得10 nM之最終濃度)的組合溶液建立該分析。建立除添加5 μl分析緩衝液代替添加測試選擇性化合物外含有全部上文分析組分的總結合對照孔。建立除添加5 μl分析緩衝液代替添加His-Fab外含有全部包括於總結合對照孔之分析組分的陰性結合對照孔。取決於特定實驗，測試化合物系列滴度係1/2或1/3及在化合物特異性基礎上優化最高最終分析化合物濃度。在獨立實驗中，於Fab特異性基礎上優化生物素化之替格瑞洛及His-Fab之濃度。對於在先導分離期結束時研究之四個Fab(TICA0010、TICA0049、TICA0053及TICA0072)而言，使用之最終分析試劑濃度在下文表1中列出：
表1 抗體ID [抗體](nM) [生物素化之替格瑞洛](nM)
TICA0039 16.0 139.9
TICA0049 16.0 37.9
TICA0053 16.0 70.7
TICA0072 8.0 17.6
對於在先導優化期結束時研究之兩個Fab(TICA0162及TICA0212)而言，在兩種情況下使用之最終分析試劑濃度係5 nM生物素化之替格瑞洛及1 nM His Fab。若干用於此等實驗中之測試選擇性化合物溶於100%DMSO及在分析信號中媒劑有關之降低可開始出現於大於約1%DMSO之濃度。為能夠隨後標準化數據以校正任何此媒劑有關之效應，單獨DMSO之平行滴定係包含於多數實驗中，其中最終分析DMSO濃度反映彼等測試化合物連續稀釋之濃度。在建立程序末期，在Envision板讀取器上使用標準讀數方案讀取前，於室溫培養該分析3小時。
對於隨後數據分析而言，首先將原始665 nm及620 nM計數轉化為665 nm/620 nm比值，其隨後用於根據分析1中陳述之等式計算ΔF%值。用於計算ΔF%之陰性比值係由陰性結合對照孔衍生。ΔF值%隨後用於根據下文等式計算特異性結合值%：
特異性結合(%)={(樣品ΔF-陰性結合ΔF)/(總結合ΔF-陰性結合ΔF)}x100
對於標準計算特異性結合%而言，總結合ΔF係獲自含有分析之全部上文提及之組分但不具有任何競爭性測試化合物的總結合對照孔。
在其中應用DMSO標準化之彼等實驗中，基本上根據上文等式計算DMSO標準化之特異性結合%，除非此情況下總結合ΔF係獲自含有全部在總結合對照孔中之組分以及DMSO濃度等於其在相關樣品孔中之濃度的孔。
獲自此類型初始實驗之結果在表2中匯總。在研究之四個初始Fab之三者(TICA0010、TICA0049及TICA0053)中，化合物坎格瑞洛顯示競爭性抑制Fab結合至生物素化之替格瑞洛。在TICA0049情況下，潘妥拉唑及利奈唑胺均顯示部分競爭性抑制。如在表2中顯示，TICA0072 Fab不顯示抑制十二種化合物中之十一者，而潘妥拉唑顯示弱部分抑制。
在此第一系列實驗中測試之全部四個Fab中觀察到抑制未經修飾之替格瑞洛及TAM，IC50
值落於0.1 μM至0.5 μM範圍。亦檢測四個Fab之二者(TICA0049及TICA0072)對TIM之競爭性抑制，然而，IC50
值＞50 μM，表明與未經修飾之替格瑞洛及TAM相比大幅度降低之對TIM親和性。基於在表2中結果，確定TICA0072具有最有利選擇性曲線。基於在剩餘三個Fab中此Fab顯示最少結合至坎格瑞洛之標準確定TICA0049為潛在備用。
表2.用於抑制生物素化之替格瑞洛結合至各個測試Fab之12個測試化合物(圖4)各者及未經修飾之替格瑞洛、TAM及TIM的相對IC50
值。 用於先導 his-Fab 之各種化合物之 IC50
(uM)
化合物 TICA0010 TICA0049 TICA0053 TICA0072
非諾貝特 NI NI NI NI
尼伐地平 NI NI NI NI
西洛他唑 NI NI NI NI
布拉地新 NI NI NI NI
瑞加德松 NI NI NI NI
環噻嗪 NI NI NI NI
賽扶寧 NI NI NI NI
洛伐他汀 NI NI NI NI
利奈唑胺 NI 467.1 NI NI
辛伐他汀 NI NI NI NI
坎格瑞洛 102.4 207.9 17.5 NI
潘妥拉唑 NI 263.7 NI 498.0
替格瑞洛 0.122 0.257 0.109 0.113
TAM 0.124 0.412 0.134 0.299
TIM NI 53.4 NI 54.4
NI係無抑制。
在第二系列實驗中產生TICA0049及TICA0072 Fab之進一步選擇性數據，其中連同替格瑞洛、TAM及TIM及若干腺苷相關化合物重新測試在圖 4
中列出之一組十二個化合物之四者。此處，對在表2中較早研究之細化，設計實驗以使得百分數特異性結合值標準化以校正任何由於DMSO之非特異性媒劑有關效應。描繪獲自此第二系列實驗之數據的實例係在圖 5
中顯示，連同在表3中製表之IC50
值。
表3.在競爭性結合選擇性研究中在圖 4
中列出之十二個化合物之子組四者、替格瑞洛、TAM、TIM及若干腺苷族化合物的實例IC50
結果(DMSO標準化) 化合物 TICA0049 TICA0072
腺苷 NI NI
ADP NI NI
2MeS ADP NI NI
ATP NI NI
2 MeS ATP NI NI
布拉地新 864.9 NI
利奈唑胺 902.0 NI
坎格瑞洛 188.4 NI
潘妥拉唑 243.5 1546.0
替格瑞洛 0.368 0.356
TAM 0.366 0.483
TIM 74.8 119.5
如同較早實驗，TICA0072顯示是最有利選擇性曲線，而在測試之四個化合物中僅潘妥拉唑顯示微弱部分抑制(＞1500 μM)。在TICA0049情況下，坎格瑞洛及潘妥拉唑觀察到顯著抑制而利奈唑胺及布拉地新觀察到微弱抑制。應注意，針對某些分析化合物在第一與第二系列實驗間絕對IC50
值之細微差異基本上不改變總體結論。此等差異可能源自下列事實組合：將DMSO標準化整併至第二系列實驗的事實，並與在若干情況下吾人嘗試測量非常弱抑制之事實。
對於TICA0049及TICA0072兩者而言，針對替格瑞洛及TAM測得之IC50
值在0.3 μM至0.5 μM之範圍，而針對TIM之IC50
值在大於2對數值，分別高於74.8 μM及119.5 μM。對於TICA0049及TICA0072 Fab二者而言，在腺苷、ADP、ATP及後二化合物之甲基硫代衍生物中觀察到輕微抑制，然而此僅於測試之最高化合物濃度下檢測到且不視為顯著。
由此得出結論，TICA0072係唯一視為對替格瑞洛及TAM具有特異性之Fab。實例 4 ：抗替格瑞洛 /TAM Fab 之親和性測量
使用生物層干涉計在Octet Red384上測定上文產生之抗替格瑞洛Fab之親和性。對於親和性測量而言，抗替格瑞洛Fab抗體在分析緩衝液(PBS，Tween20 0.05%，BSA 0.02%)中稀釋至2x最終分析濃度之濃度，例如200 nM。在Greiner聚丙烯96孔板中製備10點2倍連續稀釋之替格瑞洛。隨後將等體積(例如，70 μL加70 μL)經稀釋之抗體及游離替格瑞洛轉移至第二Greiner聚丙烯板。藉由吸量管混合該樣品，使用板蓋覆蓋及容許於室溫平衡3至5天。在平衡後，將60 μL抗體/替格瑞洛滴定以一式兩份轉移至384孔黑色傾斜底聚丙烯板。在分析緩衝液中將生物素化之替格瑞洛稀釋至250 nM及添加至384孔分析板之前2欄之交替孔，剩餘孔僅含有分析緩衝液。將鏈黴抗生物素蛋白生物感測器在分析緩衝液中預先浸漬至少10分鐘。樣品板及生物感測器隨後放置在OctetRed384之載物台上。
全部分析於室溫下進行。在分析緩衝液中基線平衡60 sec後，將生物素化之替格瑞洛裝載在鏈黴抗生物素蛋白生物感測器上300 sec，接著在分析緩衝液600 sec以建立新基線。該抗體/替格瑞洛混合物隨後容許與生物素化之替格瑞洛感測器表面締合30至600 sec，取決於使用之抗體濃度而定。使用OctetRed數據分析軟體分析所得締合期數據。針對各個樣品，將信號對齊至基線及減去參考感測器信號(無抗體對照)，隨後使用KinExA n型曲線分析軟體導出用於分析之數據。使用Constant Partner分析，測定抗替格瑞洛Fab抗體之平衡KD。數據顯示Fab TICA0072及TICA0049分別具有7.4 nM及11.6 nM之替格瑞洛親和性(表4)。
表4.抗替格瑞洛Fab之平衡親和性分析 抗體 ID 半抗原 平衡 KD 95% 置信區間
TICA0072 替格瑞洛 7.43 nM 1.75-21.46 nM
TICA0049 替格瑞洛 11.6 nM 1.7-66.5 nM
實例 5 ：優化抗替格瑞洛 /TAM 抗體 TICA0072
使用基於親和性之噬菌體選擇優化抗體TICA0072。使用如描述之標準分子生物技術(Clackson及Lowman 2004 Practical Approach Series 266)藉由可變重(VH)互補決定區(CDR)1、2或3或可變輕(VL)鏈CDR1、2或3之寡核苷酸導向突變創建由先導scFv序列衍生之大型scFv庫。該庫經歷基於親和性噬菌體呈現選擇以選擇具有對替格瑞洛及TAM較高親和性之變異體。簡言之，在具有減低生物素化之替格瑞洛濃度(典型實例應係20 nM至20 pM，經四個選擇循環後)的溶液中培養scFv噬菌體粒子，基本上如先前描述(Thompson等人，1996 J Mol Biol. 256 (1):77-88)。製備獲自CDR靶向選擇輸出之代表性數個個別scFv的粗製含有scFv之周質提取物及在HTRF®
抗原決定基競爭性分析形式中篩選，該分析形式設計為篩選相對於TICA0072之親和性之改良。
簡言之，為篩選具有改良親和性之scFv及Fab變異體，HTRF®
抗原決定基競爭性分析係藉由測試scFv變異體基於在親本TICA0072 IgG與生物素化之替格瑞洛間之交互作用的競爭性實施。此分析用作初級單點HTS以篩選粗製周質抽提物scFv樣品以及作為多點二級譜化(profiling)分析以測量純化之scFv及Fab變異對親本TICA0072之IC50
值之改良。雖然抗原決定基競爭性分析(諸如本文描述之一者)並非一般用於測定絕對親和性值，但是此等分析可用作基於親和性之HTS的基礎。此外，純化之scFv/Fab變異體(相對於親本scFv/Fab)之IC50
倍數改良可提供良好親和性總倍數增益之指示及可代表在先導優化活動中親和性分級scFv/Fab變異體之有效途徑。本文描述之TICA0072親本IgG基抗原決定基競爭性分析之形式在下文列出：
銪標識之鏈黴抗生物素蛋白：生物素化之替格瑞洛；TICA0072 IgG：XL665
標識之抗人類Fc抗體
在包括DPBS pH 7.4(Gibco 14190-086)、KF(VWR 103444T)(0.4M)及Tween 20(Sigma P9416)(0.05%)之緩衝液中於黑色淺孔384孔分析板(Corning/Costar 3676)中進行此分析。對於粗製周質scFv變異體之單點測試而言，使用10 μl分析體積，然而當在多點二級IC50
譜化分析中測試純化scFv及Fab時，使用20 μl分析體積。
對於單點HTS，藉由添加3 μl TICA0072 IgG(53.3 nM以獲得16 nM最終濃度)、2 μl粗製周質提取物scFv樣品、2.5 μl生物素化之替格瑞洛(8 nM以獲得2 nM最終濃度)及2.5 μl含有銪標識之鏈黴抗生物素蛋白(CisBio 610SAKLB)(3 nM，用於0.75 nM之最終分析濃度)及XL665
標識之抗人類Fc抗體(CisBio 61HFCXLB)(30 nM以獲得7.5 nM之最終分析濃度)的組合溶液建立該分析。親本TICA0072粗製周質scFv用作基準點及HTS經組態以識別提供相對於親本改良抑制之變異體。總結合對照孔含有除添加2 μl分析緩衝液代替scFv樣品外全部分析組分。陰性結合對照孔含有除添加3 μl分析緩衝液代替TICA0072 IgG外全部總結合對照孔組分。
對於純化scFv/Fab變異體之多點IC50
測試而言，藉由添加5 μl TICA0072 IgG(53.3 nM以獲得16 nM最終濃度)、5 μl 1/3滴定之純化測試scFv或Fab變異體、5 μl生物素化之替格瑞洛(8 nM以獲得2 nM最終濃度(scFv譜化)、4 nM以獲得1 nM最終分析濃度(Fab譜化))及5 μl含有銪標識之鏈黴抗生物素蛋白(CisBio 610SAKLB)(3 nM，用於0.75 nM最終分析濃度)及XL665
標識之抗人類Fc抗體(CisBio 61HFCXLB)(30 nM以獲得7.5 nM之最終分析濃度)之組合溶液建立該分析。在全部實驗中純化之親本TICA0072(scFv或Fab)係用作基準使得使用優化之變異體(scFv或Fab)測得之IC50
之改良可表示為相對親本TICA0072之倍數改良。總結合對照孔含有除添加5 μl分析緩衝液代替純化scFv或Fab樣品處外全部分析組分。陰性結合對照孔含有除添加5 μl分析緩衝液代替TICA0072 IgG外總結合對照孔之全部組分。
在分析之單點HTS及多點IC50
譜化版本中，於室溫培養板3小時，然後使用其中於337 nm激發樣品及於620 nm及665 nm測量時差熒光發射之標準HTRF讀取方案在Envision板讀取器上讀取。
原始665 nm及620 nm計數用於根據早前分別在分析1及4中描述之等式計算ΔF(%)及特異性結合%。對於多點二級譜化實驗而言，藉由Graphpad Prism軟件使用S形劑量反應(可變斜率)曲線擬合(4參數邏輯方程)測定IC50
值。
在篩選中識別之Hit，即當與親本TICA0072 scFv相比時顯示顯著改良之抑制效應之scFv變異體，經歷DNA測序，及隨後將獲自可變重CDR1、CDR2或CDR3及可變輕庫CDR1、CDR2或CDR3輸出之獨特變異體製備為純化scFv及在相同分析中重新測試以測定濃度反應IC50
曲線。隨後將顯示最多改良之IC50
值之scFv變異體製備為Fab及在下文描述之第二代抗原決定基競爭性分析中測試。用於篩選 / 分級最高親和性 Fab 之第二代抗原決定基競爭性分析
為在先導優化活動末期非常高親和性純化Fab間有效區分，實施另一HTRF®
抗原決定基競爭性分析，然而在此情況下該分析係基於結合至生物素化之替格瑞洛的中間產物親和性優化之TICA0072世系IgG(TICA0159)而不是親本TICA0072 IgG之競爭性抑制。此分析僅用於多點IC50
譜化形式(而不是HTS形式)及基本上與以在分析5(上文)中針對在親本TICA0072 IgG基抗原決定基競爭性分析中純化Fab變異體之多點IC50
譜化給出之方法相同地進行。唯一差異係在分析建立適當點使用TICS0159 IgG代替TICS0072 IgG但是相同16 nM最終分析濃度。在全部其他態樣中，此分析精確地如上文在針對純化Fab之多點二級譜化版本中描述進行。
與TICA0072基抗原決定基競爭性分析所能達到相比，使用部分優化之抗體TICA0159替代TICA0072之第二代分析能夠更有效區分及分級最高親和性Fab。
最多改良之VH識別為CDR3變異體TICA0162。最多改良之VL識別為CDR3變異體TICA0152。為產生進一步親和性改良，使用標準分子生物技術將源自經改良抗體之不同CDR組合為新Fab。自此重組操作，TICA0162與TICS0152之組合產生具有進一步改良之抗原決定基競爭曲線的新Fab TICA0212。在第二代抗原決定基競爭性分析中描繪之TICA0072、TICA0152、TICA0162及TICA0212 Fab競爭曲線係在圖 6
中顯示，測得之IC50
值在表5中。TICA0212顯示IC50
之相對於親本TICA0072 Fab之約2對數改良。
表5：在第二代抗原決定基競爭性分析中列出之優化抗體格瑞洛Fab之IC50
數據。 Fab IC50
(nM) 改良倍數
TICA0072 1714.0 0
TICA0152 73.5 23.3
TICA0162 16.3 105.2
TICA0212 12.0 142.8
實例 6 ：優化之抗替格瑞洛 /TAM Fab 之親和性測量
使用KinExA3200測定在實例5中產生之抗替格瑞洛/TAM Fab之親和性。對於KinExA親和性測量而言，首先藉由使其與1 mg鏈黴抗生物素蛋白在50 mM NaHCO3
中反應整夜製備珠粒(600 mg吖内酯珠粒)。在使用2種變化之Tris緩衝液(1M Tris pH 8.7，10 mg/mL BSA)阻斷之後，該鏈黴抗生物素蛋白塗覆之珠粒以8 mL總體積再懸浮。使用PBS徹底清洗珠粒(1.33 mL，等於100 mg初始乾燥吖內酯珠粒)，隨後容許在1 mL PBS中結合至約2.5 μg生物素-替格瑞洛10分鐘並且偶爾攪拌。使用PBS清洗所得生物素-替格瑞洛塗覆之珠粒，隨後再懸浮於含有0.1%BSA及0.02%NaH3
之50 mL PBS中及於室溫儲存直至轉移至KinExA珠粒小瓶。
基本上如先前描述之方法進行抗體/替格瑞洛樣品製備。在分析緩衝液(PBS，Tween20 0.05%，BSA 0.02%，0.02% NaN3
)中將抗替格瑞洛Fab抗體稀釋至2倍於最終分析濃度之濃度(例如200 nM)。在Falcon 50 mL聚丙烯管中製備10點2倍連續稀釋之替格瑞洛。隨後將等體積(例如5 mL加5 Ml)稀釋之抗體及游離替格瑞洛轉移至第二Falcon聚丙烯管中。藉由吸量管混合樣品及容許於室溫平衡3至5天。平衡後，將該樣品管轉移至KinExA 3200以用於分析。
全部分析於室溫進行。取樣抗體/替格瑞洛混合物及容許與生物素-替格瑞洛珠粒混合同時清洗掉未結合之游離替格瑞洛。隨後使用DyLight649標識之小鼠抗人類重及輕鏈抗體檢測結合之抗體。樣品體積(300至1300 μL)及注射時間(90至120 s)隨濃度變化，及每樣品變化2至3珠粒。使用KinExA n型曲線分析軟體分析數據。使用Constant Partner分析測定抗替格瑞洛Fab抗體之平衡KD。
如先前實例，在20倍過量濃度之未經修飾替格瑞洛或TAM存在下容許Fab於室溫平衡。將生物素化之替格瑞洛裝載在鏈黴抗生物素蛋白塗覆之珠粒表面。平衡後，容許剩餘游離抗體結合至生物素化之替格瑞洛。隨後使用DyLight649標識之小鼠抗人類重及輕鏈檢測抗體檢測結合之抗體。使用至少三個不同混合濃度之抗體製備游離替格瑞洛或TAM之滴定以產生在表觀KD中具有至少10倍偏移之獨立滴定曲線。使用KinExA Pro n型曲線分析軟體分析數據以測定游離替格瑞洛或TAM之平衡KD。Fab TICA0212具有約20 pM之未經修飾替格瑞洛及TAM之親和性(表6)。根據平衡數據，TICA0212證實結合至替格瑞洛及TAM之相當高親和性(~20 pM)。
表6：抗替格瑞洛/TAM Fab之平衡親和性分析 抗體 ID CDR3 序列 (SEQ ID NO) 半抗原 平衡 KD 95% C. I.
TICA0072 VH GSHLY99
DFW100b
SASHPPNDALAI (35)
VL GTW91
D92
I93
S94
LSAGL (40) 替格瑞洛 7.4 nM 1.8-21.5 nM
TICA0152 VH GSHLYDFWSASHPPNDALAI (65)
VL GTWLYDRAV
GL (70) 替格瑞洛 43.17 pM 2.8-119.2 pM
TICA0162 VH GSFD
YY
FWSASHPPNDALAI (55)
VL GTWDISLSAGL (60) 替格瑞洛 162.48 pM 125.4-206.3 pM
TICA0212
(MEDI2452)
VH GSFD
YY
FWSASHPPNDALAI (75)
VL GTWLYDRAV
GL (80) 替格瑞洛 19.6 pM 13.0-28.7 pM
TAM 19.7 pM 4.9-44.7 pM
TIM ~ 20 nM
加粗
自親本TICA0072之序列殘基之變化，及kabat號碼係針對在TICA0072中特定殘基進行確定。實例 7 ：抗替格瑞洛 /TAM Fab TICA0162 及 TICA0212 之特異性
如在實例3中測試Fab TICA0162及TICA0212之特異性及經DMSO標準化。TICA0162及TICA0212之全部可用選擇性數據之匯總表係包含於表7。此外，描繪獲自其中連同替格瑞洛、TAM、TIM及之數個腺苷族化合物測試的在圖 4
中列出之十二個化合物之五者之實驗的數據之實例係在圖 7
中顯示。
表7：除替格瑞洛、TAM、TIM及腺苷族化合物外之十二個結構相關化合物用於抑制生物素化之替格瑞洛結合至TICA0162及TICA0212 Fab的相對IC50
值。 用於先導 his-Fab 之各種化合物之 IC50
(uM)
化合物 TICA0162 TICA0212
非諾貝特 NI NI
尼伐地平 NI NI
西洛他唑 NI(n=2) NI(n=2)
布拉地新 NI NI
瑞加德松 NI(n=2) NI(n=2)
環噻嗪 ＞1000(n=2) NI(n=2)
賽扶寧 NI NI
洛伐他汀 NI NI
利奈唑胺 NI NI
辛伐他汀 NI(n=2) NI(n=2)
坎格瑞洛 NI NI
潘妥拉唑 ＞1000(n=2) NI(n=2)
腺苷 NI NI
ADP NI NI
2MeS-APD NI NI
ATP NI NI
2MeS-ATP NI NI
替格瑞洛 0.023(n=2) 0.035(n=2)
TAM 0.032(n=2) 0.031(n=2)
TIM 19.8(n=2) 28.8(n=2)
NI係無抑制。
如由數據闡明，TICA0212(NEDI2452)具有實質上對替格瑞洛及TAM相當結合特異性，及較弱結合至TIM。此外，MEDI2452/TICA0212不顯示明顯結合至任何其他結構相關藥物或腺苷相關化合物。實例 8 ：蛋白質結晶學
將具有C末端his-標籤之TICA0072在PBS中濃縮至9 mg/ml。藉由添加溶於DMSO之1 mM替格瑞洛達成複合物形成。在使用沉滴式蒸汽擴散方法進行結晶試驗前，於室溫培養該複合物2小時。使用3種商業篩進行廣泛篩選及獲得若干命中物。由Morpheus®
(Molecular Dimensions，UK)命中物網格優化獲得最佳繞射晶體。藉由混合等體積TICA0072-替格瑞洛複合物與12.8% PEG 3350、12.8% PEG 1000、12.8% MPD、1.7% 1,6-己二醇、1.7% 1-丁醇、1.7% 1,2-丙二醇、1.7% 2-丙醇、1.7% 1,4丁二醇、1.7% 1,3-丙二醇、25 mM咪唑、25 mM二甲胂酸鈉、25 mM MES及25 mM Bis-Tris pH 6.5之貯槽溶液於20℃生長該用於結構測定之晶體。在液氮中急驟冰凍該晶體而不添加任何低溫保護劑。
以約15 mg/ml之濃度含在PBS中之TICA0212/MEDI2452與替格瑞洛混合至1 mM之濃度及在進行廣泛篩選前於室溫培養2小時。未獲得自發命中物。藉由在30 μl孔溶液中粉碎數個晶體製備獲自TICA0072-替格瑞洛晶體之晶種及用於MMS(微晶種基質篩選)至廣泛商業篩。獲得若干命中物，但用於結構測定之晶體於20℃在20%甘油、20% PEG 4000、10% 2-丙醇、0.1 M NaCl及0.5 M NaAcetate pH 4.6條件下生長。使用0.2 μl蛋白質、0.18 μl孔溶液及0.02 μl晶種原液製備該滴劑。在液氮中急驟冰凍該晶體而不添加任何低溫保護劑。
於歐洲同步輻射設備(Grenoble，France)之射束線ID23-1收集數據。使用AutoProc工作流程[Vonrhein, C.等人，Acta Cryst. 2011；D67: 293-302]處理、按比例調整及進一步降低該數據，參見表8統計。對於TICA0072而言，藉由分子替代使用高解析度Fab結構(PDB 標識碼1aqk，[Faber C.等人，Immunotechnology. 1998;3:253-70])作為起始模型完成初始相。對於TICA0212/MEDI2452而言，TICA0072結構用作起始模型。使用Coot[Bricogne G.等人，(2011). BUSTER 版本2.11.4. Cambridge, United Kingdom: Global Phasing Ltd]進行模型重建及使用自動粉粹機[Emsley, P.等人，Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004，D60，2126-2132]進行細化。最終模型統計參見表8。 表 8 ：
獲自Fab替格瑞洛共晶體結構之數據收集及細化統計括號內值指最高解析度殼。描述 TICA0072 TICA0212
(MEDI2452)
PDB訪問碼
數據收集統計
輻射源 ESRF/ID23-1 ESRF/ID23-1
輻射檢測器 Pilatus Pilatus
空間群 P21 P21212
晶胞尺寸 a=41.2, b=72.6, c=67.8 β=98.9 a=69, b=173, c=42
解析度(Å) 49-1.7(1.87-1.7) 41-2.16(2.27-2.16)
觀察之反射 143002 232480
獨特反射 41888 36570
完整性(%) 97.0(95.3) 99.4 (94.7)
平均值I/σI 13.6(1.7) 10.7(1.2)
Rsym
%b 4.8(74.4) 9.7(27.7)
細化統計
解析度(Å) 49-1.7 41-2.16
蛋白質+配位體原子數量 3308 3371
溶劑原子數量 142 87
R(%)，Rfree
(%) 19.8，23.7 21.7，25.7
Wilson B(Å2
)，細化＜B＞(Å2
) 45.6，48.5 42.1，44.6
Rmsd理想鍵長度(Å) 0.008 0.010
鍵角(°) 1.10 1.25
以1.7 Å解析度測定在與替格瑞洛之複合物中TICA0072之結構 (圖10)。CDR形成高度凹曲面及替格瑞洛深***VH與VL域間界面。通常在小型半抗原中觀察到此結合類型。除VL CDR2外全部CDR直接構成替格瑞洛結合及大部分VH CDR3異常。替格瑞洛之二氟苯基位於經包括游標殘基VH Trp47、VL Phe98及VH CDR3殘基Leu100L之疏水性殘基填充之凹穴中。與替格瑞洛交互之主要殘基係VL Trp91，其涉及pi-堆疊對抗類腺苷核心及氫鍵接至於環戊基部分之核糖羥基之一。額外對類腺苷核心之交互作用由VH CDR1 His35及VH CDR3 Tyr99提供。硫丙基取代基堆疊對抗VH CDR2環之主鏈。在環戊基部分之羥基乙基取代基伸至溶劑及不產生任何與Fab之交互作用。
在親和性改良之Fab TICA0212/MEDI2452結構中，替格瑞洛結合係相似於具有全部上文提及交互保留但具有一些重要差異之TICA0072之替格瑞洛結合(圖10B)。VL CDR3突變Asp92Leu及Ser94Asp之組合破壞在VL CDR3環中之氫鍵以產生更「鬆弛」結構。新構型與關於附著環丙基-二氟苯基取代基15°傾斜之嘧啶環相關。嘧啶環之新位置使其與Fab72相比在TICA0212/MEDI2452中約0.2 Å更接近VH CDE3Tyr99。此外，VL Ile93Tyr引入產生與VH CDR3環路之交互作用的氫鍵供體，由此限定進一步結合位點。
TICA0212/MEDI2452之晶體結構顯示替格瑞洛限制於VH
與VL
界面間之深裂隙。由於晶體結構表明羥基乙基不涉及任何與TICA0212/MEDI2452之交互作用，證實經由三醯胺連接子至羥基乙基之使用生物素標識替格瑞洛之設計策略。此進一步由Fab亦以相同親和性結合缺乏羥基乙基之TAM的事實支持。TICA0212/MEDI2452顯示弱結合至缺乏環丙基-二氟苯基之TIM。在TICA0212/MEDI2452-替格瑞洛複合物中，該環丙基-二氟苯基埋於疏水凹穴底部及必須在對準具有VL
CDR3殘基Trp L91之替格瑞洛類腺苷核心時發揮關鍵結構作用。由於替格瑞洛化學起始點係ATP及其保留類腺苷核心，解毒劑特異性之關鍵屬性係證實不結合腺苷。此先導單離策略包含高產量及詳細特異性分析及藉由競爭性或直接結合分析檢測不結合腺苷。根據結構分析，可期望腺苷之嘌呤環及核糖基模擬替格瑞洛之類腺苷核心之交互作用。然而，缺乏結合可由不存在兩個顯著降低相對形狀互補性及結合交互之疏水性的疏水性R基(環丙基-二氟苯基及硫丙基)解釋。
親本TICA0072及TICA0212/MEDI2452之結構分析顯示在親和性成熟期間引入一些改變之重要性。在VL
CDR3中之突變顯示出具有特別重大影響，從而產生在TICA0212/MEDI2452中不同環路構型及額外氫鍵以限定結合凹穴。相比之下，在VH
CDR3中突變之影響係結構較不明顯。此等獲自結構之觀察係部分由含有僅在VL
CDR3(TICA0152)或VH
CDR3(TICA0162)中修飾之修飾抗體的數據確認，其與TICA0072相比分別導致200倍及50倍改良。然而，儘管VL
CDR3改變顯示具有較高影響，二組突變添加顯著改良。應注意晶體結構係複合物之靜止圖片及不能捕捉涉及結合之任何蛋白質及配位體動態。實例 9 ：在替格瑞洛或 TAM 存在下體外 TICA0212/MEDI2452 濃度依賴性恢復之血小板凝聚
在富含人類血小板之血漿(PRP)中使用光透射集合度測定法測定TICA0212/MEDI2452逆轉替格瑞洛或TAM介導之抑制ADP誘發之血小板凝聚的程度及能力。
對於體外人類PRP分析而言，從空腹健康志願者藉由頭靜脈靜脈穿刺收集血液。在將等分試樣收集至含有0.109 M檸檬酸鈉之試管前丟棄最初之2 mL血液，1+9(檸檬酸鹽+血液)，至10.9 mM之最終濃度。於240 x g離心抗凝血之人類血液15 min。小心移除PRP及轉移至乾淨小瓶中。藉由於2000 x g離心PRP 15 min製備貧血小板血漿(PPP)。藉由血小板凝聚測繪器(PAP-8E，Bio/Data Corporation，PA，USA)在PRP中評估光透射集合度(LTA)。零%凝聚定義為PRP之光透射及100%凝聚定義為PPP之光透射。
在與不同濃度TICA0212或同型物對照Fab共培養30分鐘前，使用1 μM替格瑞洛或TAM預培養PRP 1小時。藉由添加20 μM ADP引發血小板凝聚及持續記錄6 min。分析於6 min之最終凝聚(FA)程度數據。
計算提供半最大值逆轉之TICA0212/MEDI2452濃度(IC50
)。TICA0212/MEDI2452產生1 μM替格瑞洛及1 μM TAM介導之20 μM ADP-誘發之血小板凝聚抑制之濃度依賴性逆轉，計算之平均(n=5)IC50
值分別為0.64及0.78 μM(圖 8
)。當於1:1條件(1 μM TICA0212/MEDI2452:1 μM替格瑞洛或TAM)評估時，逆轉之平均程度分別係78%及62%。同型物對照Fab不引發明顯替格瑞洛及TAM ADP誘發之血小板凝聚的逆轉。例如，在30 min培養後，同型對照Fab分別產生-3%及2%替格瑞洛及TAM逆轉。
由此，數據顯示TICA0212/MEDI2452可以濃度依賴方式體外逆轉替格瑞洛及TAM介導之ADP誘發血小板凝聚之抑制。當在1:1實驗設置評估時(可預測當TICA0212/MEDI2452以1:1化學計量比結合至替格瑞洛或TAM時)，達成最大及幾乎完全之逆轉效應。實例 10 ：在替格瑞洛或 TAM 存在下 TICA0212/MEDI2452 有效及快速恢復體外血小板凝聚
在富含人類血小板血漿(PRP)中使用如在實例8中之光透射集合度測定法測定TICA0212/MEDI2452逆轉替格瑞洛或TAM之起效時間。在添加1 μM TICA0212/MEDI2452及共培養5、10、15、30及60分鐘，或添加同型物對照Fab共培養30分鐘前，使用1 μM替格瑞洛或TAM預培養PRP 1小時。藉由添加20 μM ADP引發血小板凝聚及持續記錄6 min。分析於6 min之最終凝聚(FA)程度數據。
TICA0212/MEDI2452產生無關共培養時間之相似對替格瑞洛介導之抑制之逆轉程度，在5、10、15、30及60分鐘後平均(n=3)逆轉程度分別係85%、69%、74%、80%、及81%。相似地，在5、10、15、30及60分鐘後由TICA0212/MEDI2452引發之平均(n=3)TAM逆轉程度分別係53%、56%、58%、69%、74%。TICA0212/MEDI2452快速及有效逆轉替格瑞洛及介導之ADP誘發之血小板凝聚之抑制。在與同型物對照Fab共培養30 min後不存在逆轉(平均(n=3)-2%)。根據此數據，當以1:1實驗設置評估時TICA0212/MEDI2452顯示快速及有效逆轉替格瑞洛及TAM介導之ADP誘發凝聚之抑制。實例 11 ：在投藥給經替格瑞洛治療之小鼠後 TICA0212/MEDI2452 有效及快速恢復體內血小板凝聚
在靜脈內(i.v.)投與小鼠替格瑞洛後，體外測定在全血(阻抗集合度測定法)中TICA0212/MEDI2452介導之逆轉替格瑞洛介導之ADP誘發的血小板凝聚之抑制的起效速度及程度。經5分鐘靜脈內呈1200 μg/kg大劑量投與小鼠替格瑞洛，接著15分鐘持續輸注30 μg/kg/min。在終止替格瑞洛輸注後，當測量之替格瑞洛血漿暴露為平均1.4 μM時，給予小鼠250 mg/kg TICA0212/MEDI2452之靜脈大劑量。在5、30、及60 min TICA0212/MEDI2452投藥後，殺死小鼠及收集血樣。在此研究中測量ADP誘發之凝聚反應6 min及數據表示為在隨時間記錄之凝聚單位(AU)曲線下方的平均面積(AU*min)。
在進行阻抗集合度測定法分析時，殺死小鼠及將血樣收集至7 μM水蛭素。在多板微測試單元中將血液(175 μL)添加至預加熱之NaCl2
(37℃，175 μL)及在添加12 μL ADP至6.5 μM最終濃度前混合3 min。該可棄式多板微測試單元含有攪拌棒及具有兩對分離之浸入血樣中的電極。
當添加促效劑(ADP)及藉由拌攪引發剪切時，血小板開始附著及凝聚在電極上。這導致電極上阻抗增加，其藉由多板阻抗集合度計(DynaByte，Munchen，Germany)隨時間持續記錄。
由於在替格瑞洛輸注及PBS大劑量後5、30及60 min時，平均(n=4)凝聚反應分別從432降低至2、從474至6及從494至14 AU*min(圖 9
)，所以替格瑞洛處理誘發幾乎完全之ADP誘發之凝聚抑制。由於在替格瑞洛輸注及TICA0212大劑量後5、30、及60分鐘時，平均(n=4)凝聚反應分別從2增加至147、從6至448及從14至412 AU*min(圖 9
)，所以TICA0212/MEDI2452介導之體內替格瑞洛介導之抑制之逆轉。由於平均(n=4)凝聚反應仍從6至4 AU*min未改變，在投與同型物對照後30分鐘不存在逆轉。
此數據顯示，當靜脈內大劑量給予投與至1.4 μM替格瑞洛血漿濃度(其提供完全ADP誘發凝聚之抑制)之小鼠時， TICA0212/MEDI2452可快速及有效恢復ADP誘發之血小板凝聚。實例 12 ：經替格瑞洛治療小鼠之小鼠出血
由於TICA0212/MEDI2452期望適應症之一係作為用於需要緊急手術之替格瑞洛患者的解毒劑，在設計為應在手術開始前達成完全逆轉之模擬臨床設置之小鼠出血實驗中評估TICA0212/MEDI2452。
經由持續輸注替格瑞洛(300 μg/kg/min)或媒劑20分鐘預處理小鼠。在停止輸注後，t=0，經45秒給予TICA0212/MEDI2452(600 mg/kg)或媒劑(組胺酸蔗糖緩衝液)之大劑量及於t=30分鐘藉由切斷5 mm尾尖誘發出血。使用水(2 mL/min)淋洗尾尖及在小容器中收集血液與水之混合物，其中攪拌混合該液體以增強溶血作用及建立均相溶液。當從腹部大動脈收集末端血樣時記錄於525 nm之光透射30分鐘用於血小板凝聚。光透射率可轉化為吸光度及用於計算作為在吸收曲線下面積(AUC，吸光度*s)之血液損失及藉由繪製隨時間之吸光度計算總出血時間(BT，s)。全部低於95%之透射率定義為出血。亦於替格瑞洛輸注(t=0)末期及於尾部切斷(t=30分鐘)時收集血小板凝聚及全部與游離血漿暴露樣品。
由瑞典哥德堡大學(University of Göteborg，Sweden)的動物研究倫理委員會批准研究。使用異氟醚氣體(Forene®，Abbot Scandinavia AB，Sweden)麻醉小鼠。將導管***左側頸靜脈以用於投與媒劑或藥物。體溫藉由外熱維持於38℃。
為將TICA0212/MEDI2452對血小板凝聚之效應轉化為潛在出血效應，進行預防小鼠尾部出血研究。將替格瑞洛分別融合至7.6及0.3 μM之平均總體格瑞洛及TAM血漿濃度以提供顯著藥物依賴性出血窗。在停止替格瑞洛輸注後立即經30秒投與600 mg/kg TICA0212/MEDI2452之單一大劑量。在30分鐘後，ADP誘發之凝聚全部正常化及替格瑞洛之平均游離血漿濃度從4.7 nM降低至低於0.03 nM(量化下限)。藉由尾部切斷引發出血及監測30分鐘。在經媒劑處理之小鼠中，於尾部切斷時平均總替格瑞洛及TAM血漿濃度係2.4及0.6 μM。經30分鐘總血液損失及出血時間係分別由替格瑞洛顯著(p＜0.05)加強約3.8倍及1.6倍。TICA0212/MEDI2452顯著 (p＜0.05)逆轉血液損失及相對於單獨替格瑞洛之出血時間及恢復至不顯著區別於未經替格瑞洛處理之小鼠的程度(圖12)。在此預防設置下，TICA0212/MEDI2452使替格瑞洛依賴性出血正常化。從體外模型轉移至此體內模型之30分鐘起效時間證實TICA0212/MEDI2452可使血液損失及出血時間恢復至未經替格瑞洛處理之小鼠的血液損失及出血時間。實例 13 ：替格瑞洛及 TAM 之總血漿濃度
在具有EDTA抗凝血劑之試管中收集血樣及在10000 x g下於室溫離心5 min於以製備血漿。藉由蛋白質沉澱及使用串聯式質譜法之液相層析(LC-MS/MS)測定替格瑞洛及TAM之血漿濃度，如在具有如下偏差之已公開方法[Sillén H.等人，J Chromatogr B Analyt Technol Biomed Life Sci 2010;878:2299–306]中描述。血漿(50 μL)係使用180 μl內標物(D7-ZD6140)在乙腈中進行蛋白質沉澱。該液相層析系統及質譜儀係獲自Waters之Acquity Ultra Performance LC耦合Xevo TQ-S質譜儀。在Aquity UPLC® BEH C18管柱上(2.1 x 50mm，粒度1.7 µm)達成層析分離。採用陰性電噴霧電離。溶離劑A係含有10 mmol/L乙酸銨之水，pH 5及溶離劑B係含有10 mmol/L乙酸銨之乙腈。注射體積係1至5 μL及該分析梯度起始於4%B，在1.5 min內增加至95%，維持直至2.3 min，然後在2.4 min內返回初始條件，接著再次平衡0.3 min。在此分析中不使用品質對照樣品。定量下限(LLOQ)係0.005 μmol/L及校正範圍係0.005至15.0 μM。
藉由添加1%甲酸(FA:樣品，1:5) ，接著蛋白質沉澱及如上文描述之LC-MS/MS 測定在TICA0212/MEDI2452存在下替格瑞洛及TAM之總血漿濃度。將甲酸添加至樣品以促進替格瑞洛及TICA0212/MEDI2452之解離。實例 14 ：替格瑞洛游離血漿濃度
基於先前公開之方法[Sillén H.等人，J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Aug 1;879(23):2315-22]優化該方法。將容許具有低於6至8 kDa質量之分子通過的透析膜(Spectrum Laboratories，Inc)在ELGA水中浸泡10至15 min及放置在半透析板間(室內製備)。在透析板一側添加130 μL血漿及將130 μL磷酸鹽緩衝液(pH7.0)添加至相對側。在兩側之孔使用蓋子封住及將鋁板放置在該板各側頂部以避免滲漏。該板隨後垂直放置在於100 rpm/min在37℃之迴轉式振盪器上持續24 h。藉由將50 μL滯留物從血漿側及75 μL透析物從緩衝液側轉移至蛋白質LoBind PCR清潔96深孔板來終止透析，該深孔板含有150 μL在乙腈中之各自75 μL內標物(D7-ZD6140)。混合該板1 min及隨後，1500 x g於4℃離心20 min。在離心後，轉移50 μL獲自沉澱滯留物之上清液及使用50 μL ELGA H2
O稀釋，然後LC-MS/MS(Acquity Ultra Performance LC 耦合Xevo TQ-S質譜儀，Waters)分析。在該分析中不使用品質對照樣品。滯留物校準範圍係0.4至1000 nmol/L及透析物校準範圍係0.003至50 nmol/L。在透析物中替格瑞洛之LLOQ中係0.03 nmol/L。
下文表9提供上文實例中描述之例示性抗體之概述及用於說明本文揭示技術之某些實施例。
表9.抗體/scFv/Fab序列之匯總 抗體 Ref 序列 序列
TICA0010 Vh DNA (SEQ ID NO:1) gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aacagagtac gacctgcaac ggcctttcgg gtttgacttc tggggcaagg ggacaatggt caccgtctcg agt
TICA0010 Vh (SEQ ID NO:2) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMS
WVRQAPGKGLEWVSAISGSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCATEYDLQRPFGFDF
WGKGTMVTVSS
TICA0010 Vh CDR1 (SEQ ID NO:3) SYAMS
TICA0010 Vh CDR2 (SEQ ID NO:4) AISGSGGSTYYADSVKG
TICA0010 Vh CDR3 (SEQ ID NO:5) EYDLQRPFGFDF
TICA0010 Vl DNA (SEQ ID NO:6) tcctatgtgc tgactcagcc accctcagcg tctggggccc ccgggcagag ggctaccatc tcctgctctg gaagcagctc caacatcgga agtaatcttg tgaactggta ccaacaattc ccaggagagg cccccaagct cctcatcttt agtgacaatc aacgaccctc aggggtccct gaccgattct ctggctccag gtctggcacc tcagcctccc tggccatcag tgggctccag tccgaggatg aggctgatta ttactgtgca acgtgggatg acagactgga tggttatgtg gtattcggcg gagggaccaa gctgaccgtc cta
TICA0010 Vl (SEQ ID NO:7) SYVLTQPPSASGAPGQRATISCSGSSSNIGSNLVN
WYQQFPGEAPKLLIFSDNQRPS
GVPDRFSGSRSGTSASLAISGLQSEDEADYYCATWDDRLDGYVV
FGGGTKLTVL
TICA0010 Vl CDR1 (SEQ ID NO:8) SGSSSNIGSNLVN
TICA0010 Vl CDR2 (SEQ ID NO:9) SDNQRPS
TICA0010 Vl CDR3 (SEQ ID NO:10) ATWDDRLDGYVV
TICA0049 Vh DNA (SEQ ID NO:11) caggtacagc tgcagcagtc aggggctgag gtgaagaagc ctggggcctc agtgaaggtt tcctgcaagg cttctggata caccttcatt acctatggta ttcactgggt gcgccaggcc cccggacaag ggcttgagtg gatgggatgg atcgaccccg ggcatggtta cacaaaatat tcacagaagt tccagggcag agtcaccatt accagggaca catccgcgag cacagcctac atggagatga gcagcctcag atctgaagac acggctgtgt attactgtgc gagagcggac ctgggtgact actggggccg gggaaccctg gtcaccgtct cgagt
TICA0049 Vh (SEQ ID NO:12) QVQLQQSGAEVKKPGASVKVSCKASGYTFITYGIH
WVRQAPGQGLEWMGWIDPGHGYTKYSQKFQG
RVTITRDTSASTAYMEMSSLRSEDTAVYYCARADLGDY
WGRGTLVTVSS
TICA0049 Vh CDR1 (SEQ ID NO:13) TYGIH
TICA0049 Vh CDR2 (SEQ ID NO:14) WIDPGHGYTKYSQKFQG
TICA0049 Vh CDR3 (SEQ ID NO:15) ADLGDY
TICA0049 Vl DNA (SEQ ID NO:16) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcagctc caacattggg aagaattatg tttcctggtt ccagcagctc ccaggtacag cccccaaact cctcatttat gacaatcata agcgaccctc agggattcct gaccgattct ctgcctccaa gtctggcacg tcagccaccc tggtcatctc cggtctccag actggggacg aggcccatta ttactgcgga acatgggata ccagactgag tgctggggtg ttcggcggag ggaccaaggt caccgtccta
TICA0049 Vl (SEQ ID NO:17) QSVVTQPPSVSAAPGQKVTISCSGSSSNIGKNYVS
WFQQLPGTAPKLLIYDNHKRPS
GIPDRFSASKSGTSATLVISGLQTGDEAHYYCGTWDTRLSAGV
FGGGTKVTVL
TICA0049 Vl CDR1 (SEQ ID NO:18) SGSSSNIGKNYVS
TICA0049 Vl CDR2 (SEQ ID NO:19) DNHKRPS
TICA0049 Vl CDR3 (SEQ ID NO:20) GTWDTRLSAGV
TICA0053 Vh DNA (SEQ ID NO:21) gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgg ccatgatagt agtggttact cctactcctt tgacttctgg gggcggggga ccacggtcac cgtctcgagt
TICA0053 Vh (SEQ ID NO:22) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMS
WVRQAPGKGLEWVSAISGSGGSTYYADSVKG
RFTISRDNSKNTLYLQMNSLRAEDTAVYYCGHDSSGYSYSFDF
WGRGTTVTVSS
TICA0053 Vh CDR1 (SEQ ID NO:23) SYAMS
TICA0053 Vh CDR2 (SEQ ID NO:24) AISGSGGSTYYADSVKG
TICA0053 Vh CDR3 (SEQ ID NO:25) DSSGYSYSFDF
TICA0053 Vl DNA (SEQ ID NO:26) cagtctgtgt tgacgcagcc gccctcagcg tctgggaccc ccgggcagag ggtcaccatc tcttgttctg gcaacatctc caacatcgga agtaacactg tcaactggta tcaacacgtc ccaggagcgg cccccagact cctcatctat gttaatgatc agcggccgtc aggggtccct gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag tctgaagatg aggctgatta ttactgtgca acgtgggatg acaccctgaa tggaggggtc ttcggcggag ggaccaagct gaccgtccta
TICA0053 Vl (SEQ ID NO:27) QSVLTQPPSASGTPGQRVTISCSGNISNIGSNTVN
WYQHVPGAAPRLLIYVNDQRPS
GVPDRFSGSKSGTSASLAISGLQSEDEADYYCATWDDTLNGGV
FGGGTKLTVL
TICA0053 Vl CDR1 (SEQ ID NO:28) SGNISNIGSNTVN
TICA0053 Vl CDR2 (SEQ ID NO:29) VNDQRPS
TICA0053 Vl CDR3 (SEQ ID NO:30) ATWDDTLNGGV
TICA0072 Vh DNA (SEQ ID NO:31) caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagagggtcc catctttacg atttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc caaggaaccc tggtcaccgt ctcgagt
TICA0072 Vh (SEQ ID NO:32) QVQLQESGAEVKKPGSSVRVSCKASGGTFDSYSIH
WVRQAPGQGLEWMGGIIPAFGTLSSAQDFQA
RVTISADKSTSTAYMELSGLRSEDTAVYYCARGSHLYDFWSASHPPNDALAI
WGQGTLVTVSS
TICA0072 Vh CDR1 (SEQ ID NO:33) SYSIH
TICA0072 Vh CDR2 (SEQ ID NO:34) GIIPAFGTLSSAQDFQA
TICA0072 Vh CDR3 (SEQ ID NO:35) GSHLYDFWSASHPPNDALAI
TICA0072 Vl DNA (SEQ ID NO:36) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag gctggggacg aggccgatta ttactgcggg acatgggata tcagcctgag cgctggcttg ttcggcggag ggaccaaggt caccgtccta
TICA0072 Vl (SEQ ID NO:37) QSVVTQPPSVSAAPGQKVTISCSGSNSDIGNNYVS
WYQQLPGTAPKLLIYDNNKRPS
GIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWDISLSAGL
FGGGTKVTVL
TICA0072 Vl CDR1 (SEQ ID NO:38) SGSNSDIGNNYVS
TICA0072 Vl CDR2 (SEQ ID NO:39) DNNKRPS
TICA0072 Vl CDR3 (SEQ ID NO:40) GTWDISLSAGL
TICA0159 Vh DNA (SEQ ID NO:41) caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagagggagc ttcgactaca ggttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc caaggaaccc tggtcaccgt ctcgagt
TICA0159 Vh (SEQ ID NO:42) QVQLQESGAEVKKPGSSVRVSCKASGGTFDSYSIH
WVRQAPGQGLEWMGGIIPAFGTLSSAQDFQA
RVTISADKSTSTAYMELSGLRSEDTAVYYCARGSFDYRFWSASHPPNDALAI
WGQGTLVTVSS
TICA0159 Vh CDR1 (SEQ ID NO:43) SYSIH
TICA0159 Vh CDR2 (SEQ ID NO:44) GIIPAFGTLSSAQDFQA
TICA0159 Vh CDR3 (SEQ ID NO:45) GSFDYRFWSASHPPNDALAI
TICA0159 Vl DNA (SEQ ID NO:46) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag gctggggacg aggccgatta ttactgcggg acatgggata tcagcctgag cgctggcttg ttcggcggag ggaccaaggt caccgtccta
TICA0159 Vl (SEQ ID NO:47) QSVVTQPPSVSAAPGQKVTISCSGSNSDIGNNYVS
WYQQLPGTAPKLLIYDNNKRPS
GIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWDISLSAGL
FGGGTKVTVL
TICA0159 Vl CDR1 (SEQ ID NO:48) SGSNSDIGNNYVS
TICA0159 Vl CDR2 (SEQ ID NO:49) DNNKRPS
TICA0159 Vl CDR3 (SEQ ID NO:50) GTWDISLSAGL
TICA0162 Vh DNA (SEQ ID NO:51) caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagaggctcc ttcgactact acttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc caaggaaccc tggtcaccgt ctcgagt
TICA0162 Vh (SEQ ID NO:52) QVQLQESGAEVKKPGSSVRVSCKASGGTFDSYSIH
WVRQAPGQGLEWMGGIIPAFGTLSSAQDFQA
RVTISADKSTSTAYMELSGLRSEDTAVYYCARGSFDYYFWSASHPPNDALAI
WGQGTLVTVSS
TICA0162 Vh CDR1 (SEQ ID NO:53) SYSIH
TICA0162 Vh CDR2 (SEQ ID NO:54) GIIPAFGTLSSAQDFQA
TICA0162 Vh CDR3 (SEQ ID NO:55) GSFDYYFWSASHPPNDALAI
TICA0162 Vl DNA (SEQ ID NO:56) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag gctggggacg aggccgatta ttactgcggg acatgggata tcagcctgag cgctggcttg ttcggcggag ggaccaaggt caccgtccta
TICA0162 Vl (SEQ ID NO:57) QSVVTQPPSVSAAPGQKVTISCSGSNSDIGNNYVS
WYQQLPGTAPKLLIYDNNKRPS
GIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWDISLSAGL
FGGGTKVTVL
TICA0162 Vl CDR1 (SEQ ID NO:58) SGSNSDIGNNYVS
TICA0162 Vl CDR2 (SEQ ID NO:59) DNNKRPS
TICA0162 Vl CDR3 (SEQ ID NO:60) GTWDISLSAGL
TICA0152 Vh DNA (SEQ ID NO:61) caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagagggtcc catctttacg atttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc caaggaaccc tggtcaccgt ctcgagt
TICA0152 Vh (SEQ ID NO:62) QVQLQESGAEVKKPGSSVRVSCKASGGTFDSYSIH
WVRQAPGQGLEWMGGIIPAFGTLSSAQDFQA
RVTISADKSTSTAYMELSGLRSEDTAVYYCARGSHLYDFWSASHPPNDALAI
WGQGTLVTVSS
TICA0152 Vh CDR1 (SEQ ID NO:63) SYSIH
TICA0152 Vh CDR2 (SEQ ID NO:64) GIIPAFGTLSSAQDFQA
TICA0152 Vh CDR3 (SEQ ID NO:65) GSHLYDFWSASHPPNDALAI
TICA0152 Vl DNA (SEQ ID NO:66) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag gctggggacg aggccgatta ttactgcggg acatggctgt acgaccgggc cgtcggcttg ttcggcggag ggaccaaggt caccgtccta
TICA0152 Vl (SEQ ID NO:67) QSVVTQPPSVSAAPGQKVTISCSGSNSDIGNNYVS
WYQQLPGTAPKLLIYDNNKRPS
GIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWLYDRAVGL
FGGGTKVTVL
TICA0152 Vl CDR1 (SEQ ID NO:68) SGSNSDIGNNYVS
TICA0152 Vl CDR2 (SEQ ID NO:69) DNNKRPS
TICA0152 Vl CDR3 (SEQ ID NO:70) GTWLYDRAVGL
TICA0212/MEDI2452 Vh DNA (SEQ ID NO:71) caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagaggctcc ttcgactact acttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc caaggaaccc tggtcaccgt ctcgagt
TICA0212/MEDI2452 Vh (SEQ ID NO:72) QVQLQESGAEVKKPGSSVRVSCKASGGTFDSYSIH
WVRQAPGQGLEWMGGIIPAFGTLSSAQDFQA
RVTISADKSTSTAYMELSGLRSEDTAVYYCARGSFDYYFWSASHPPNDALAI
WGQGTLVTVSS
TICA0212/MEDI2452 Vh CDR1 (SEQ ID NO:73) SYSIH
TICA0212/MEDI2452 Vh CDR2 (SEQ ID NO:74) GIIPAFGTLSSAQDFQA
TICA0212/MEDI2452 Vh CDR3 (SEQ ID NO:75) GSFDYYFWSASHPPNDALAI
TICA0212/MEDI2452 Vl DNA (SEQ ID NO:76) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag gctggggacg aggccgatta ttactgcggg acatggctgt acgaccgggc cgtcggcttg ttcggcggag ggaccaaggt caccgtccta
TICA0212/MEDI2452 Vl (SEQ ID NO:77) QSVVTQPPSVSAAPGQKVTISCSGSNSDIGNNYVS
WYQQLPGTAPKLLIYDNNKRPS
GIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWLYDRAVGL
FGGGTKVTVL
TICA0212/MEDI2452 Vl CDR1 (SEQ ID NO:78) SGSNSDIGNNYVS
TICA0212/MEDI2452 Vl CDR2 (SEQ ID NO:79) DNNKRPS
TICA0212/MEDI2452 Vl CDR3 (SEQ ID NO:80) GTWLYDRAVGL
參考文獻及以引用之方式併入
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本文提及之所有公開案及專利申請案其全文係以引用的方式併入文中，其程度就如同個別公開案或專利申請案特定地且個別地以引用的方式併入般。
當討論本發明之特殊態樣時，上文說明書係闡述性及並非限制性。對熟習此項技術者而言，在審查此說明書及下文申請專利範圍後，本發明之眾多變化將變得明顯。本發明之全部範疇應參考申請專利範圍以及其等效物之全部範疇、及說明書，以及此等變化而確定。 electronically Submit sequence list Of refer to
This application incorporates by reference the Sequence Listing in computer readable form (CRF), which is a text file and series titled "Ticagrelor_SeqList_ST25.TXT" created on October 1, 2014, which is filed with this application. 33,900 bytes in size.
Before proceeding to describe the present invention in greater detail, it is to be understood that this invention is not limited to particular compositions or method steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd Edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd Edition, 1999, Academic Press; and Oxford Dictionary Of Biochemistry And Molecular Biology, Revised Edition, 2000, Oxford University Press, provides a general dictionary of many terms used in the present invention for those skilled in the art.
Amino acids herein may be cited by their commonly known three-letter symbols or single-letter symbols as suggested by the IUPAC-IUB Biochemical Nomenclature Committee. Likewise, nucleotides may refer to their generally accepted word codes.
Unless otherwise specified, the numbering of amino acids in the variable domains, complementarity determining regions (CDRs), and framework regions (FRs) of antibodies follows the Kabat definitions, as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed, Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortened, or inserted, FRs or CDRs of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and an inserted residue after residue 82 of heavy chain FR (eg, residue according to Kabat) bases 82a, 82b, and 82c, etc.). The Kabat numbering of residues in a given antibody can be determined by alignment in regions of homology to antibody sequences with "standard" Kabat numbering sequences. Maximal alignment of framework residues often requires the insertion of "spacer" residues in the numbering system for Fv domains. Furthermore, the identification of specific individual residues numbered at any given Kabat site may vary from antibody chain to antibody chain due to interspecies or allelic differences.
As used herein, the terms "antibody" and "antibody, etc.," also referred to as immunoglobulins, include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, as many as formed from at least two different epitope-binding fragments Specific antibodies (eg, multispecific antibodies, eg, PCT Publication WO2009018386, PCT Application No. PCT/US2012/045229, which are incorporated herein by reference in their entirety), biMabs, human antibodies, humanized antibodies, Camelized antibody, single chain Fv (scFv), single chain antibody, single domain antibody, domain antibody, Fab fragment, F(ab')2
Fragments, antibody fragments exhibiting the desired biological activity (eg, antigen-binding moieties), disulfide-bonded Fvs (dsFv), and anti-idiotypic (anti-Id) antibodies (including, eg, anti-Id antibodies to antibodies of the invention) , intrabodies, and epitope-binding fragments of any of the foregoing. In certain embodiments provided herein, the antibodies refer to active binding fragments of antibodies, ie, molecules that contain at least one antigen-binding site, such as, for example, scFvs and Fabs. Antibodies also include peptides fused to antibodies or portions thereof, such as proteins fused to an Fc domain. Immunoglobulin molecules can be of any isotype (eg, IgG, IgE, IgM, IgD, IgA, and IgY), sub-isotype (eg, IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2), or allotype (eg, Gm , eg, G1m (f, z, a, or x), G2m (n), G3m (g, b, or c), Am, Em, and Km (1, 2, or 3)). Antibodies can be derived from any mammal (including, but not limited to, humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc.), or other animals such as birds (eg, chickens).
As used herein, C1
-C6
Alkyl refers to straight and branched chain alkyl groups having one to six carbon atoms, and includes methyl, ethyl, propyl, n-butyl, isobutyl, pentyl, isopentyl, neopentyl, and hexyl .
As used herein, C1
-C6
Alkoxy refers to an alkyl group having one oxygen in the group as indicated above. In some embodiments, the oxygen atom is located at the position where the substituent is attached to the core structure (ie, the ring structure).
As used herein, C1
-C6
Alkylthio refers to an alkyl group having one sulfur in the group as indicated above. In some embodiments, the sulfur atom is located at the position where the substituent is attached to the core structure (ie, the ring structure).
As used herein, C1
-C6
Alkanol refers to an alkyl group having a hydroxyl group at the end of the substituent structure as indicated above.
As used herein, C3
-C6
Cycloalkyl refers to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.
As used herein, the "substituted" C3
-C6
Cycloalkyl and C1
-C6
Alkyl refers to the alkyl and cycloalkyl groups discussed above substituted on at least one carbon atom by an aryl group also substituted with 1 to 3 halogen atoms.
As used herein, "ticagrelor" refers to the reversible P2Y12
Inhibitor ((1S,2S,3R,5S)-3-[7-{[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino}-5-(propyl Thio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol ) and have the following chemical structures:.
As used herein, "ticagrelor active metabolite" or "TAM" refers to the major active metabolite of ticagrelor, also known as AR-C124910XX, a reversible P2Y12
Inhibitors have the following chemical structures:.
As used herein, "ticagrelor inactive metabolite" or "TIM" refers to the inactive metabolite of ticagrelor, also known as AR-C133913XX, and having the following chemical structure:.Antibody
In a general sense, the present invention provides the combined formula (Ia
) novel antibodies of cyclopentyltriazolopyrimidine compounds:
in
R1
Department selected from C1
-C6
Alkoxy and C1
-C6
A group consisting of alkylthio groups;
R2
The system is selected from H, C1
-C6
Alkyl, substituted C1
-C6
Alkyl, C3
-C6
Cycloalkyl, and substituted C3
-C6
the group consisting of cycloalkyl groups; and
R3
The system is selected from H, C1
-C6
Alkyl, C1
-C6
Alkoxy, and C1
-C6
A group of alkanols.
In particular embodiments, the antibody specifically binds a compound selected from the group consisting of:ticagrelor;ticagrelor active metabolite (TAM); andTicagrelor inactive metabolite (TIM).
In particular aspects, the invention provides an antibody that binds to ticagrelor and TAM having any one or more of the following characteristics, including high binding specificity, high binding affinity, rapid onset of action, and rapid Offsetting (eg, allowing for continuation or co-administration of treatment including ticagrelor as appropriate).
In some embodiments, the antibody binds to ticagrelor and neutralizes the antiplatelet aggregation activity of ticagrelor and TAM, thereby restoring ADP-induced platelet aggregation in the presence of ticagrelor and TAM.
In some embodiments, the antibody half-life in the subject is approximately equal to the half-life of ticagrelor and TAM. In some embodiments, the half-life of the antibody is about 4 to 24 hours (eg, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23 or 24 hours). In some embodiments, the antibody half-life is about 4 to 12 hours (eg, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours).
In some embodiments, the antibody provides rapid onset of activity. For example, in embodiments, the time to onset of action of the antibody, or the time to neutralize ticagrelor and TAM-mediated platelet inhibition, is about 15 to 120 minutes, or about 15 to 60 minutes. In some embodiments, the onset time is less than 60 minutes.
In some embodiments, the antibody has a PK/PD profile that provides rapid activity cancellation, such that, eg, a subject administered the antibody can resume prescribed ticagrelor treatment. In some embodiments, a subject receiving an antibody disclosed herein (eg, by i.v. infusion) may receive or restart ticagrelor treatment within twenty-four hours of administration of the antibody.
As discussed and exemplified in certain embodiments herein, the antibody binds ticagrelor or a metabolite thereof and does not bind to other structurally related compounds, or can be administered with ticagrelor as a co-therapy compound. For example, suitably the antibody is non-inhibitory selected from the group consisting of fenofibrate, nilvadipine, cilostazol, bradysine, regadesone, cyclothiazide, severin, lovastatin, linezole Activity of compounds of the group consisting of amine, simvastatin, cangrelor, pantoprazole, adenosine, adenosine diphosphate, adenosine triphosphate, 2-MeS adenosine diphosphate, and 2-MeS adenosine triphosphate.
Antibodies described herein can include antigen-binding fragments (such as Fab, F(ab') containing only selected portions of the antibody molecule2
, Fab', scFv, di-scFv, sdAb fragments) and can be used as diagnostic or therapeutic agents. In addition, specific residues in the variable domains can be altered to improve the binding specificity and/or stability of antibodies and antibody fragments. Other residues not directly involved in antigen binding can be substituted to "humanize" regions of the non-human antibody and reduce the immunogenicity of the antibody.
In certain aspects, the antibody is a Fab fragment, eg, an antibody Fab comprising a variable light chain (VL), a constant light chain (CL), a variable heavy chain (VH), and a constant heavy chain portion (CH1 ). Fragments or recombinantly produced antigen-binding fragments. Optionally, the light and heavy chains of the Fab may be interconnected via one or several disulfide bonds, such as, for example, via a suitable antibody hinge region. As described herein, the Fab binds to an epitope of an orally active agent compound of the cyclopentyltriazolopyrimidine class. In some embodiments, the Fab binds to ticagrelor or a metabolite thereof.
In certain aspects, Fabs can be derived from or based on antibody sequences, such as conventional murine, humanized or human antibodies. In certain aspects, a Fab can be derived from or based on one or several scFvs, such as scFvs screened and derived from a library. In these embodiments, the Fab derived from or based on the sequence of a known antibody or scFv retains one or several functional activities of the known antibody (eg, retains at least 80% or more (80%, 85%, 90%) , 95%, 97%, 98%, 99% or 100%) functional activity). For example, in certain aspects, the Fab retains one or more of antigen (eg, ticagrelor) affinity, inhibitory activity, and/or selectivity of the antibody or scFv.
Although Fab fragments can include sequences that bind to epitopes of cyclopentyltriazolopyrimidine, in certain embodiments, the Fab binds to ticagrelor. In some aspects, the Fab binds to the active metabolite of ticagrelor. In certain aspects, the Fab can bind to both ticagrelor and the active metabolite of ticagrelor.
In some embodiments, a Fab can include a combination of CDR regions obtained from different antibodies that bind to ticagrelor or its active metabolite.
In certain aspects, the Fab includes a light chain portion (VL) included in SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:47 The amino acid sequence set forth in any of ID NO:57, SEQ ID NO:67, and SEQ ID NO:77. In other embodiments, the Fab comprises a light chain portion comprising the amino acid sequence set forth in any one of SEQ ID NO:57, SEQ ID NO:67, and SEQ ID NO:77. In certain aspects, the Fab includes a heavy chain portion (VH) included in SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 12 The amino acid set forth in any one of ID NO:52, SEQ ID NO:62, and SEQ ID NO:72. In other embodiments, the Fab includes a heavy chain portion that includes the amino acid sequence set forth in any one of SEQ ID NO:52, SEQ ID NO:62, and SEQ ID NO:72. In certain aspects, a Fab consists of a nucleotide sequence encoding the light chain portion (VL) and a nucleotide sequence encoding the heavy chain portion (VH), eg, included in SEQ ID NO: 1, SEQ ID NO: 11 , the nucleotide sequence of the nucleic acid sequence set forth in SEQ ID NO:21, SEQ ID NO:31, SEQ ID NO:41, SEQ ID NO:51, SEQ ID NO:61, or SEQ ID NO:71; and Included in SEQ ID NO:6, SEQ ID NO:16, SEQ ID NO:16, SEQ ID NO:16, SEQ ID NO:16, SEQ ID NO:16, SEQ ID NO:16, or SEQ ID NO:76 The nucleotide sequence codes for the nucleic acid sequences listed in .
In certain aspects, the antibody can be an scFv. It will be appreciated that scFv encompasses polypeptide chains comprising a variable heavy chain domain (VH) linked to a variable light chain domain (VL) via a flexible polypeptide linker. In some aspects, the polypeptide linker between VH and VL includes a protease cleavage site. The VH and VL domains of the scFv can be derived from the same or different antibodies. In some aspects, the VH or VL of the scFv can include one or more CDRs that bind to the target of interest, while the remainder of the VH or VL domain is derived from a different antibody or is synthesized. In some aspects, the scFv includes at least one CDR of an antibody (eg, an antibody having binding activity to bind to ticagrelor or a metabolite thereof). In some aspects, the scFv includes at least two CDRs of a given antibody. In some aspects, the scFv includes at least three CDRs of a given antibody. In some aspects, the scFv includes at least four CDRs of a given antibody. In some aspects, the scFv includes at least five CDRs of a given antibody. In some aspects, the scFv includes at least six CDRs of a given antibody.
Several approaches can be used alone or in combination to improve the stability of scFv molecules. One method that can be used alone or in combination with one or several other methods is to engineer the length and/or composition of the linker connecting the scFv domains to stabilize the scFv portion.
Another potential method that can be used alone or in combination with one or more of the other methods described herein is by introducing (also known as modifying or mutating) at least two amino acid substituents into the VH and/or VL domains of the scFv To promote disulfide bond formation (see, for example, Brinkmann et al., 1993, PNAS, 90:7538-42; Zhu et al., 1997, Prot. Sci. 6:781-8; Reiter et al., 1994, Biochem. 33: 5451-9; Reiter et al., 1996, Nature 14: 1239-45; Luo et al., 1995, J. Biochem. 118: 825-31; Young et al., 1995, FEBS Let. 377: 135-9; Glockshuber et al. Human, 1990, Biochem. 29:1362-7).
In certain aspects, a mutation is introduced into each of the VH and VL domains of the scFv to facilitate the formation of interchain disulfide bonds between the VH and VL domains upon scFv expression. In another aspect, the two mutations are introduced into the same domain of the chain. In certain aspects, the two mutations are introduced into different strands. In certain aspects, multiple pairs of two mutations are introduced to promote multiple disulfide bond formation. In certain aspects, cysteine is introduced to facilitate disulfide bond formation. Exemplary amino acids that can be mutated to cysteine include amino acids 43, 44, 45, 46, 47, 103, 104, 105, and 106 of VH2 and amino acids 42, 43, 44, 45 of VL2 , 46, 98, 99, 100, and 101. The previous numbering was based on Kabat numbering, which identified positions that were only related to VH2 and VL2 of the scFv (and not related to the position of the amino acid in the full-length antibody sequence). Exemplary combinations of amino acid moieties that can be mutated to cysteine residues include: VH44-VL100, VH105-VL43, VH105-VL42, VH44-VL101, VH106-VL43, VH104-VL43, VH44-VL99, VH45- VL98, VH46-VL98, VH103-VL43, VH103-VL44, and VH103-VL45. In some aspects, amino acid 44 of VH and amino acid 100 of VL are mutated to cysteine.
Yet another potential method that can be used alone or in combination with one or more of the other methods described herein is the selection of the order of scFv domains. In certain aspects, the orientation of the VH domain relative to the VL domain is optimized for stability purposes. In certain aspects, the scFv is oriented at the VH-linker-VL. In certain aspects, the scFv is oriented at the VL-linker-VH.
An additional method, which can be used alone or in combination with one or more of the methods described herein, is to introduce one or several stabilizing mutations by mutating one or several surface residues of the scFv. In some aspects, one, two, three, four, five, six, or more than six residues are mutated in one or both of the VH and/or VL domains of the scFv. In certain aspects, the alteration occurs only in the VH domain of the scFv. In certain aspects, the alteration occurs only in the VL domain of the scFv. In certain aspects, the changes occur in both the VH and VL domains of the scFv. The same amount of changes can occur in each domain or a different amount of changes can occur in each domain. In certain aspects, one or more alterations result from conservative amino acid substitutions of residues present in the unmodified parent scFv. In other aspects, one or more changes are obtained from non-conservative amino acid substitutions of residues present in the unmodified parent scFv. When several substitutions are made, each substitution is independently a conservative or non-conservative substitution in one or both of the VH or VL domains of the scFv. In certain aspects, all substitutions are conservative substitutions. In some aspects, all substitutions are non-conservative. In certain aspects, at least one of the substitutions is conservative. In certain aspects, at least one of the substitutions is non-conservative.
Yet another method, which can be used alone or in combination with one or more of the additional methods described herein, is to introduce one or several substitutions to match the The most common residues at that particular position of the consensus sequence of the VH and/or VL domain of an antibody are known, screened, and/or recognized. In certain aspects, substitutions are introduced at one or both, two, three, four, five, six, or more than six positions of the VH domain and/or VL domain of the scFv. The same amount of changes can occur in each domain or a different amount of changes can occur in each domain. In certain aspects, one or more changes in the sequence that match a given consensus sequence result from conservative amino acid substitutions of residues present in the unmodified VH and/or VL sequences. In other aspects, the one or more changes represent non-conservative amino acid substitutions obtained from residues present in unmodified VH and/or VL sequences. When multiple substitutions are made, each substitution is independently conservative and non-conservative in either or both of the VH or VL domains of the scFv. In certain aspects, all of the substitutions are conservative substitutions. In certain aspects, all substitutions are non-conservative substitutions. In certain aspects, at least one of the substitutions is conservative. In certain aspects, at least one of the substitutions is non-conservative.
It should be noted that any of the modifications described for modifying or stabilizing the scFv portion can be used to modify the Fab portion. For example, the variable domains of Fabs can be modified to improve stability, antigen binding, and the like. In addition, the Fab or scFv portion can be modified to reduce immunogenicity.
In certain aspects, the antibody can be an scFv that includes a variable light chain portion (VL) included in SEQ ID NO: 7, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 7 The amino acid sequence set forth in any one of ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:67, and SEQ ID NO:77. In other embodiments, the scFv comprises a light chain portion comprising the amino acid sequence set forth in any one of SEQ ID NO:57, SEQ ID NO:67, and SEQ ID NO:77. In certain aspects, the scFv includes a heavy chain portion (VH) included in SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:32, SEQ ID NO:42, SEQ ID NO:42, The amino acid set forth in any one of ID NO:52, SEQ ID NO:62, and SEQ ID NO:72. In other embodiments, the scFv comprises a heavy chain portion comprising the amino acid sequence set forth in any one of SEQ ID NO:52, SEQ ID NO:62, and SEQ ID NO:72.
The antibodies disclosed herein may further include one or more linker polypeptides. The linker can interconnect the heavy and light chain domains (scFv) or link the antibody or antigen-binding fragment thereof to another agent, such as a label, Fc domain, or the like. Linker lengths and sequences can vary and are generally well known in the art.
The serum half-life of an antibody comprising an Fc region can be increased by increasing the binding affinity of the Fc region for FcRn. As used herein, the term "antibody half-life" means the pharmacokinetic property of an antibody which is a measure of the average survival time of the antibody molecule after its administration. Antibody half-life can be expressed as the elimination of 50% of a known amount of immunoglobulin from a patient (or other mammal) or its specific compartment (e.g., measured in serum, i.e., circulating half-life, or in other tissues) time. Half-life can vary from one immunoglobulin or class of immunoglobulins to another. In general, increasing the half-life of an antibody results in an increase in the mean residence time (MRT) in the circulation of the antibody to which it is administered.
Increasing the half-life may allow for a reduction in the amount of agent administered to a patient as well as a reduction in the frequency of administration. To increase the serum half-life of an antibody, we can incorporate salvage receptor binding epitopes into the antibody (antibody fragments in particular), eg, as described in US Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (eg, IgGl, IgG2, IgG3, or IgG4) responsible for increasing the in vivo serum half-life of the IgG molecule. Alternatively, antibodies of the invention with increased half-life can be generated by modifying amino acid residues determined to be involved in the interaction between the Fc and the FcRn receptor (see, eg, US Pat. Nos. 6,821,505 and 7,083,784; and WO 09/058492 ). Furthermore, the half-life of the antibodies of the invention can be increased by conjugation to PEG or albumin by techniques widely employed in the art.
Antibodies within the scope of the present invention can be identified by any of the structural and/or functional characteristics identified herein. For example, antibodies can be screened for specific binding characteristics (eg, kappaoff
, KD
, IC50
, specificity/selectivity for ticagrelor and ticagrelor metabolites).Logos, Conjugates and Parts
Antibodies of the invention can be conjugated to labels for diagnostic and other analytical purposes in which the antibody and/or its target can be detected. Labels include, but are not limited to, chromophores, fluorophores, fluorescent proteins, phosphorescent dyes, tandem dyes, particles, haptens, enzymes, and radioisotopes.
In certain aspects, the antibody is conjugated to a fluorophore. The choice of fluorophore attached to the antibody will determine the absorption and fluorescence emission properties of the conjugated antibody. Physical properties useful for fluorophore labeling of antibodies and antibody-binding ligands include, but are not limited to, spectral properties (absorption, emission, and Stokes shifts), fluorescence intensity, decay period, polarization, and rate of light decolorization , or a combination thereof. All of these physical properties can be used to distinguish one fluorophore from another, and thus allow for multiplex analysis. Other desirable properties of fluorescent labels may include cell permeability and low toxicity, eg, if antibody labeling is performed in cells or model organisms (eg, living animals).
In certain aspects, the enzyme is labeled and conjugated to the antibody. Enzymes are desirably labeled because of the achievable amplification of the detectable signal resulting in enhanced assay sensitivity. The enzyme itself produces no detectable response, but when contacted with an appropriate substrate serves to destroy the substrate so that the converted substrate produces a fluorescent, colorimetric or luminescent signal. Since one enzyme can cause the conversion of multiple substrates to a detectable signal on the labeling reagent, the enzyme amplifies the detectable signal. Enzyme substrates are chosen to yield preferably measurable products, eg, colorimetric assays, fluorescence or chemiluminescence. Such substrates are widely used in the art and are well known to those skilled in the art and include, for example, oxidoreductases such as horseradish peroxidase and substrates such as 3,3'-diaminobenzidine (DAB ); phosphatases such as acid phosphatases, bases and substrates such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP); glycosidases such as β-galactosidase, β-glucuronidase or β-glucosidases and substrates such as 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal); additional enzymes include hydrolases such as cholinesterases and peptides Enzymes, oxidases such as glucose oxidase and cytochrome oxidase, and reductases for which suitable substrates are well known.
Chemiluminescence-producing enzymes and their appropriate substrates are suitable for some assays. These include, but are not limited to, native and recombinant forms of luciferase and aequorin. Chemiluminescence-producing substrates for phosphatases, glycosidases, and oxidases, such as those containing stabilized dioxetane, luminol, isoluminol, and acridium esters, are additionally useful.
In another aspect, haptens such as biotin are also used as labels. Biotin is useful because it can act on enzymatic systems to further amplify the detectable signal, and it can be used as a label for affinity chromatography for separation purposes. For detection purposes, enzyme conjugates with biotin affinity, such as avidin-HRP, are used. The peroxidase substrate is then added to generate a detectable signal.
Haptens also include hormones, natural and synthetic drugs, pollutants, allergens, effector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and and many more.
In certain aspects, fluorescent proteins can be conjugated to antibodies as labels. Examples of fluorescent proteins include green fluorescent protein (GFP) and phycochrome proteins and derivatives thereof. The fluorescent proteins (specifically phycochrome proteins) are particularly useful as labeling reagents for generating tandem dye labels. For the purpose of obtaining larger Stokes shifts, these tandem dyes include fluorescent proteins and fluorophores, where the emission spectrum is further shifted from the wavelength of the absorption spectrum of the fluorescent protein.
In certain embodiments, the label is a radioisotope. Examples of suitable radioactive substances include, but are not limited to, iodine (121
I.123
I.125
I.131
I), carbon (14
C), sulfur (35
S), tritium (3
H), indium (111
In,112
In,113
mIn,115
mIn), 鎝(99
Tc,99
mTc), thallium (201
Ti), gallium (68
Ga,67
Ga), palladium (103
Pd), molybdenum (99
Mo), Xenon (135
Xe), fluorine (18
F),153
SM,177
Lu,159
Gd,149
Pm,140
La,175
Yb,166
Ho,90
Y.47
Sc,186
Re,188
Re,142
Pr,105
Rh and97
Ru.
In some aspects, a drug can be conjugated to the antibody. For example, antibodies including scFvs can be conjugated to drugs for the treatment of cardiovascular disease and/or acute coronary heart disease.
In certain features, drugs and other molecules can be targeted to antibodies via site-specific conjugation. For example, the antibody may include a cysteine engineered domain (including cysteine engineered into a binding unit and/or an Fc domain) that generates free thiol groups for conjugation reactions. In certain aspects, the antibody is engineered to incorporate specific conjugation sites. Nucleic acid molecules encoding antibodies
The present invention provides nucleic acid molecules encoding antibodies or antigen-binding fragments thereof. One aspect of the present invention provides nucleic acid molecules encoding any of the antibodies specifically described herein. The nucleic acid molecule can encode the heavy and/or light chain variable regions of the antibody.
In some aspects, the antibody is a Fab or scFv, wherein the nucleic acid portion encoding the Fab or scFv includes a nucleotide sequence encoding a VL domain and a nucleotide sequence encoding a VH, and wherein the nucleotide sequence encoding the VL domain is The nucleotide sequence encoding the VH domain is optionally linked via a nucleotide sequence encoding a flexible polypeptide linker.
Another aspect provides a host cell transformed with any of the nucleic acid molecules as described herein. In another aspect of the invention, there is provided a host cell comprising a vector comprising a nucleic acid molecule as described herein. In one aspect, the host cell can include several vectors.
The present invention designs nucleic acid molecules encoding any of the antibodies of the invention, as well as the light or heavy chains of such antibodies. For example, the present designs include encoding one or more of SEQ ID NO: 2, SEQ ID NO: 12, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 42, SEQ ID NO: 52, SEQ ID NO: :62, SEQ ID NO:72, SEQ ID NO:7, SEQ ID NO:17, SEQ ID NO:27, SEQ ID NO:37, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:67 , and the nucleic acid molecule of the nucleotide sequence of SEQ ID NO:77. The invention further designs nucleic acid molecules encoding any of the antibodies of the invention that further comprise additional regions (eg, Fc or modified Fc). In some embodiments, the nucleic acid molecules can be selected from one or more of SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO :26, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:61, SEQ ID NO:66 , SEQ ID NO:71, or SEQ ID NO:76. In other embodiments, the present invention provides a vector comprising one or more selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6, EQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:46, SEQ ID NO:51, SEQ ID NO:56, SEQ ID NO:61, SEQ ID The nucleic acid molecule of NO:66, SEQ ID NO:71, or SEQ ID NO:76. make antibodies, Fab and scFv method
The present invention provides methods of making the antibodies and fragments thereof described herein. In some aspects, antigen-binding fragments of antibodies that recognize the specific epitopes of ticagrelor and ticagrelor and/or TAM disclosed herein can be generated by any technique known to those skilled in the art. For example, enzymes such as papain (for making Fab fragments) or pepsin (for making F(ab') can be used2
Fragments), production of Fab and F(ab') from antibodies by protease cleavage of immunoglobulin molecules2
Fragment. In addition, antibodies including scFvs and Fabs as described herein can be generated using a variety of phage display methods known in the art.
In general, in phage display methods, functional antibody domains are presented on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, DNA sequences encoding the VH and VL domains are amplified from animal cDNA libraries (eg, human or mammalian cDNA libraries of lymphoid tissue). The DNA encoding the VH and VL domains was recombined by PCR with the scFv linker and cloned into a phagemid vector. The vector was electroporated in E. coli and E. coli was infected with helper phage. Phage used in these methods can be filamentous phage including fd and M13 and the VH and VL domains can be recombinantly fused to phage gene III or gene VIII. Phage expressing antigen binding domains bound to ticagrelor and/or TAM can be selected or determined from antigens, eg, using labeled antigens or antigens bound or captured to solid surfaces or beads. Similarly, binding domains that bind to antigens/haptens other than ticagrelor and/or TAM or others can be determined to be deselected. Examples of phage display methods for preparing antibodies of the invention may include those in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186 ; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology 57:191-280; PCT application PCT/GB91/O1 134; PCT Publications WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO 97/13844; and US Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, Those disclosed in Nos. 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108; the entire contents of each of which are incorporated herein by reference.
As described in the references above, following phage selection, antibody coding regions can be isolated from phage and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragments (scFv and Fab), and in any Expression in desired hosts, including mammalian cells, insect cells, plant cells, yeast, and bacteria, eg, as described below. Methods well known in the art such as those in PCT Publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12(6):864-869; Sawai et al., 1995, AJRI 34: 26-34; and Better et al., 1988, Science 240: 1041-1043 (these references are incorporated herein by reference in their entirety) using recombinant production of Fab, Fab' and F(ab ')2
Fragment technology.
In certain aspects, the nucleic acids disclosed herein are operably linked to one or more regulatory nucleotide sequences in the expression construct. The nucleic acid sequences encoding the light and heavy chains of the antibody can be cloned in the same expression vector in either orientation (eg, light chain ahead of the heavy chain or vice versa) or can be cloned in two different vectors. If the expression line is performed using one vector, the two encoded genes may have their respective genetic elements (eg, promoter, RBS, leader, stop, polyA, etc.) or they may be cloned from a single set of genetic elements, but not cis-trans sub-connected. Regulatory nucleotide sequences will generally be appropriate for the host cell used for expression. Several types of appropriate expression vectors and suitable regulatory sequences for various host cells are well known in the art. Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription initiation and termination sequences, translation initiation and termination sequences, and Enhancement or activation sequence. Constitutive or inducible promoters, as well known in the art, are designed by the present invention. The promoter can be a native promoter, or a hybrid promoter combining several promoter elements. The expression construct may be present on an episome in the cell, such as a plastid, or the expression construct may be inserted into a chromosome.
In certain aspects, the expression vector contains a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and can vary with the host cell used. In certain aspects, the invention relates to expression vectors comprising a nucleotide sequence encoding a polypeptide and operably linked to at least one regulatory sequence. Regulatory sequences are identified by techniques and selected for direct expression of the encoded polypeptide. Thus, the term regulatory sequence includes promoters, enhancers, and other expression control factors. For example, non-limiting regulatory sequences are in Goeddel;Gene Expression Technology: Methods in Enzymology
, Academic Press, San Diego, CA (1990). It will be appreciated that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed and/or the type of protein that is desired to be expressed. In addition, the replication number of the vector, the ability to control the replication number and expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
Methods of making antibodies of the invention can include, for example, culturing one or several expression vectors encoding the antibody under appropriate conditions that allow expression of the antibody to occur (e.g., a single vector encoding the heavy and light chains or variable regions thereof, or two vectors, one encoding the heavy chain and one encoding the light chain or its variable region) into the transfected host cell. The antibody can be secreted and isolated from a mixture of cells and medium containing the antibody. Alternatively, the antibody can be retained in the cytoplasmic or membrane fraction and cells harvested, lysed and protein isolated. Cell cultures include host cells, media, and other by-products. Suitable media systems for cell culture are well known in the art. The antibody can be purified from proteins, antibodies, and antigen-binding antibody fragments thereof using techniques known in the art for purification of proteins, antibodies, and antigen-binding antibody fragments thereof, including ion exchange chromatography, gel filtration chromatography, ultrafiltration, electrophoresis, and immunoaffinity purification. Isolated in cell culture medium, host cells, or both. In certain aspects, the antibody system is prepared as an antigen-binding fragment of the antibody that includes heavy and light chain variable regions (which can increase solubility and facilitate purification).
Recombinant nucleic acids can be prepared by ligating the selection gene, or a portion thereof, to a vector suitable for expression in prokaryotic cells, eukaryotic cells (yeast, avian, insect, or mammalian), or both. Expression vehicles for the preparation of recombinant polypeptides include plastids and other vectors. For example, suitable vectors include the following types of plastids: pBR322-derived plastids, pEMBL-derived plastids, pEX-derived plastids, pBTac-derived plastids for expression in prokaryotic cells such as E. coli plastids and pUC-derived plastids. In certain aspects, mammalian expression vectors contain prokaryotic sequences that facilitate propagation of the vector in bacteria, and one or more eukaryotic transcription units that are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are suitable for transfection of eukaryotic cells for mammalian expression Examples of vectors. Some of these vectors are modified with sequences obtained from bacterial plastids, such as pBR322, to facilitate replication and resistance selection in prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can be used to express proteins transiently in eukaryotic cells. Various methods for the preparation of plastids and transformed host organisms are well known in the art. For other suitable expression systems for prokaryotic and eukaryotic cells, as well as general recombination procedures, seeMolecular Cloning A Laboratory Manual
, 2nd edition, edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, it is desirable to express recombinant polypeptides by using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as pBlueBac III containing ß-gal) .
Techniques for making fusion genes are well known. Basically, ligation of various nucleic acid fragments encoding different polypeptide/antibody sequences is performed according to known techniques, using blunt or staggered end termini for ligation, restriction enzyme digestion to provide appropriate terminus, filling sticky ends as needed, base Sex phosphatase treatment to avoid undesired ligation and enzymatic ligation. In another aspect, fusion genes can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be performed using anchor primers that generate complementary overhangs between two contiguous nucleic acid fragments (which can then be annealed to generate chimeric gene sequences) (see, e.g.,Current Protocols in Molecular Biology
, edited by Ausubel et al., John Wiley & Sons: 1992).
In some aspects, expression vectors expressing any of the nucleic acids described herein can be used to express antibodies in host cells. For example, antibodies can be expressed in bacterial cells such as E. coli, insect cells (eg, using a baculovirus expression system), yeast, or mammalian cells. Other suitable host cell lines are well known to those skilled in the art.
Once the expression vector is transferred to host cells by known techniques, the transfected cells are then cultured by known techniques to produce antibodies. Thus, the present invention includes host cells containing a polynucleotide encoding an antibody or fragment thereof, operably linked to a heterologous promoter. In certain aspects, the heavy and light chains and/or the heavy and light chain variable regions can be expressed together (from the same or different vectors) in a host cell to express the entire antibody. In certain aspects, the heavy and light chains of the antibody can be expressed from a single promoter. In certain aspects, the heavy and light chains of the antibody can be expressed from multiple promoters. In certain aspects, the heavy and light chains of the antibody can be encoded on a single vector. In certain aspects, the heavy and light chains of the antibody can be encoded on multiple vectors.
Mammalian cell lines that can be used as hosts for the expression of recombinant antibodies are well known in the art and include numerous immortalized cell lines obtained from the American Type Culture Collection (ATCC), including but not limited to Chinese Hamster Ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), human epithelial kidney 293 cells, and several other cells Tie. Different host cells have characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure proper modification and processing of the expressed antibody or portion thereof. For this purpose, eukaryotic host cells with appropriate cellular machinery for processing primary transcription, glycosylation, and phosphorylation of gene products can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (which do not endogenously produce any function murine myeloid cancer cell lines of immunoglobulin chains), SP20, CRL7O3O and HsS78Bst cells. In one aspect, human cell lines developed by immortalizing human lymphocytes can be used for recombinant production of monoclonal antibodies. In one aspect, the human cell line PER.C6. (Crucell, Netherlands) can be used for recombinant production of monoclonal antibodies.
Additional cell lines that can be used as hosts for expression of recombinant antibodies include, but are not limited to, insect cells (eg, Sf21/Sf9,Trichoplusia ni
Bti-Tn5b1-4) or yeast cells (eg, Saccharomyces cerevisiae (S. cerevisiae
), Pichia (Pichia
), US 7326681; etc.), plant cells (US20080066200); and chicken cells (WO2008142124).
In certain aspects, the antibody systems of the invention are stably expressed in cell lines. Stable performance can be used for long-term, high-yield production of recombinant proteins, antibodies and antigen-binding fragments thereof. For example, cell lines stably expressing the antibody molecule can be generated. Host cells can be transformed with appropriately engineered vectors that include expression control factors (eg, promoters, enhancers, transcription termination regions, polyadenylation sites, etc.) and selectable marker genes. Following introduction of exogenous DNA, cells can be allowed to grow in fortified medium for 1 to 2 days and then switched to selective medium. The selectable marker in the recombinant plastid confers resistance to selection and allows cells to grow and form variant regions that stably integrate the plastid into their chromosomes, which can then be cloned and expanded into cell lines. Methods for producing stable cell lines in high yields are well known in the art, and reagents are generally commercially available.
In certain aspects, the antibodies of the invention are transiently expressed in cell lines. Transient transfection is a process in which nucleic acid introduced into a cell does not integrate into the genomic or chromosomal DNA of the cell. It is actually maintained in the cell as an extrachromosomal element such as an episome. The process of transcription of the nucleic acid of the episome is not affected and the protein encoded by the nucleic acid of the episome is produced.
Stably or transiently transfected cell lines are maintained under cell culture media and conditions well known in the art, resulting in the expression and production of monoclonal antibodies. In certain aspects, mammalian cell culture media are based on commercially available media formulations including, for example, DMEM or Ham's F12. In other aspects, the cell culture medium is modified to provide increases in cell growth and biological protein expression. As used herein, the terms "cell culture medium," "medium," and "medium formulation" refer to nutrient solutions that maintain, grow, proliferate, expand cells in an artificial in vitro environment outside of a multicellular organism or tissue. Cell culture media can be optimized for specific cell culture uses, including, for example, cell growth media formulated to promote cell growth, or cell production media formulated to promote recombinant protein production. The terms nutrient, ingredient, and component are used interchangeably herein to refer to the parts that make up the cell culture medium.
Once the molecule has been produced, it can be purified by any method known in the art of purifying immunoglobulin molecules or fragments thereof, for example, by chromatography (eg, ion exchange, affinity (specifically by specificity) antigenic protein A or protein G affinity), and size exclusion column chromatography), centrifugation, differential solubility, or purification by any other standard technique for purification of proteins, antibodies, and/or antibody fragments. In addition, the molecules of the invention or fragments thereof can be fused to other heterologous polypeptide sequences described herein or well known in the art to facilitate purification (referred to herein as "tags" such as histidine tags).
When recombinant techniques are used, molecules can be prepared intracellularly, in the extracellular space, or secreted directly into the culture medium. If the molecule is produced intracellularly, as a first step, particulate debris (host cells or lysed fragments) is removed, eg, by centrifugation or ultrafiltration. Carter et al.,Bio/Technology
, 10:163-167 (1992) describes a procedure for the isolation of antibodies secreted into the extracellular space of E. coli. In the case of secretion of the molecule into the culture medium, the supernatant of these expression systems is typically first concentrated using commercially available protein concentration filters (eg, Amicon or Millipore Pellicon ultrafiltration units). Protease inhibitors such as PMSF can be included in any of the foregoing steps to inhibit proteolysis and antibiotics can be included to prevent the growth of additional contaminants.
Can be purified using, for example, hydroxide apatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and/or affinity chromatography, alone or in combination with other purification steps Compositions prepared from cells. The suitability of Protein A as an affinity ligand depends on the species and isotype (if present) of any immunoglobulin Fc domain in the molecule, and will be understood by those skilled in the art. The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene can be achieved to allow faster flow rates and shorter processing times than agarose. Other protein purification techniques such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin chromatography, anion or cation exchange resins (such as polyaspartic acid columns) SEPHAROSE chromatography, chromatography Focusing, SDS-PAGE, and ammonium sulfate precipitation are also available, depending on the molecules to be recovered.
After any preliminary purification steps, mixtures including molecules of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using elution buffer at a pH between about 2.5 and 4.5, and at low salt concentrations (eg, about 0 to 0.25M salt).
Antibodies can be prepared and purified using, for example, any or a combination of the techniques listed above and/or in the Examples. Regardless of how the antibody is purified, in order to confirm the functional binding of the antibody of the invention, a binding assay can be performed (before and/or after purification). For example, a dual ELISA assay can be used. In some aspects, a first antigen (eg, ticagrelor or a competitor thereof) is coated on the wells, and antibodies bound to this antigen are immobilized to prepare it for detection. pharmaceutical formulation
In certain aspects, the present invention provides pharmaceutical compositions. Such pharmaceutical compositions may be compositions comprising nucleic acid molecules encoding antibodies. Such pharmaceutical compositions may also be compositions comprising antibodies, or combinations of antibodies, and pharmaceutically acceptable excipients. In certain aspects, the pharmaceutical compositions of the present invention are used as medicaments.
In certain aspects, the antibody or combination of antibodies (or nucleic acid molecule encoding the antibody or combination of antibodies) can be formulated as a pharmaceutical composition with pharmaceutically acceptable carriers, excipients, or stabilizers. In certain aspects, these pharmaceutical compositions are suitable for administration to humans or non-human animals via any one or more routes of administration using methods well known in the art. As will be understood by those skilled in the art, the route and/or mode of administration may vary depending on the desired result. The term "pharmaceutically acceptable carrier" refers to one or more non-toxic substances that do not interfere with the effectiveness of the active ingredient's biological activity. Such formulations may generally contain salts, buffers, preservatives, compatible carriers, and optionally other therapeutic agents. Such pharmaceutically acceptable formulations may also contain compatible solid or liquid fillers, diluents or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and/or additives that can be used in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients such as Serum albumin, gelatin, casein, salt form counterions such as sodium and the like. These and additional well-known pharmaceutical carriers, excipients and/or additives suitable for use in the formulations described herein are well known in the art, eg, as in "Remington: The Science & Practice of Pharmacy", p. 21 ed. Lippincott Williams & Wilkins, (2005), and "Physician's Desk Reference", 60th ed., Medical Economics, Montvale, N.J. (2005). A pharmaceutically acceptable carrier can be selected as appropriate for the desired or required mode of administration, solubility and/or stability.
The formulations described herein include an active agent (eg, an antibody or antibody fragment such as a Fab or scFv) at a concentration that yields w/v suitable for the desired dose. In certain aspects, the active agent is administered at about 1 mg/ml to about 200 mg/ml, about 1 mg/ml to about 100 mg/ml, about 1 mg/ml to about 50 mg/ml, or about Concentrations from 1 mg/ml to about 25 mg/ml are present in the formulation. In certain aspects, the active agent is present at a concentration of about 25 mg/ml. In certain aspects, the active agent concentration in the formulation can vary from about 0.1 to about 100% by weight. In certain aspects, the active agent concentration is in the range of 0.003 to 1.0 moles.
In one aspect, the formulations of the present invention are pyrogen-free formulations that are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined to the interior of a microorganism and that are released only when the microorganism breaks or dies. Pyrogenic substances also include fever-causing, thermostable substances (glycoproteins) derived from the outer membrane of bacteria or other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to potentially deleterious effects, even small amounts of endotoxin must be removed from solutions of medicinal products administered intravenously. The U.S. Food and Drug Administration (“FDA”) has set an upper limit of 5 endotoxin units (EU)/dose/kg body weight during one hour of intravenous drug application (The United States Pharmacopeial Convention, Pharmacopeial Forum 26(1):223 (2000)). In certain specific aspects, the endotoxin and pyrogen levels in the composition are less than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or less than 0.1 EU/mg, or less than 0.01 EU /mg, or less than 0.001 EU/mg.
When used for in vivo administration, the formulations of the present invention should be sterile. The formulations of the present invention can be sterilized by various sterilization methods, including sterile filtration, irradiation, and the like. In one aspect, the formulation is filter sterilized through a pre-sterilized 0.22 micron filter. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice (as described in "Remington: The Science & Practice of Pharmacy", 21st Ed., Lippincott Williams & Wilkins, (2005)).
The therapeutic compositions of the present invention can be formulated for particular routes of administration, such as oral, nasal, pulmonary, topical (including buccal and sublingual), rectal, vaginal and/or parenteral. As used herein, the phrases "parenteral administration" and "parenteral administration" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, Intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular , subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Formulations of the invention suitable for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The antibody may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and any desired preservatives, buffers, or propellants (US Pat. Nos. 7,378,110; 7,258,873; 7,135,180; US Publication No. 2004-0042972; and 2004-0042971).
The formulations may conveniently be presented in unit dosage form and prepared by any method known in the art of pharmacy. The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention can be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient (eg, "therapeutically effective" quantity"). The dose level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, and the particular composition employed The compounds and/or materials used in combination, the age, sex, weight, status, general health and previous drug history of the patient to be treated, and similar factors well known in the pharmaceutical art. Suitable dosages may vary from about 0.0001 to about 100 mg/kg body weight or more, eg, about 0.1, 1, 10, or 50 mg/kg body weight, with about 1 to about 10 mg/kg body weight being suitable.
It should be noted that the present invention is similarly designed to also prepare formulations suitable for diagnostic and research use. The concentration of active agent in these formulations, as well as the presence or absence of excipients and/or pyrogens, can be selected based on the particular application and intended use. use
The antibodies disclosed herein are suitable for use in therapeutic methods, including combination therapy, for neutralizing the activity of platelet activation, aggregation and degranulation inhibitors, platelet disaggregation promoters, and antithrombotic agents. Thus, the antibodies described herein have been found to be useful in several applications related to the administration of ticagrelor (including concomitant methods) and are suitable for neutralizing one or several metabolites of ticagrelor and/or ticagrelor effect of things. In these methods, the antibody optionally reversibly reduces, neutralizes, eliminates, or otherwise inhibits the activity of ticagrelor and the treatment or prevention is associated with administration of ticagrelor and/or derived from ticagrelor including Any number of effects, diseases and/or symptoms of Relorex treatment.
The antibody can be administered to patients who are to be treated or in need of treatment or prophylaxis for ticagrelor (BRILINTA) treatable and/or indicated indications including, for example, unstable angina, primary atherosclerosis Arterial thrombotic complications such as thrombotic or embolic stroke, transient ischemic attack, peripheral vascular disease, myocardial infarction with or without thrombosis, interventions due to atherosclerotic disease such as angioplasty (including Arterial complications, surgical or mechanical injury due to coronary angioplasty (PTCA), endarterectomy, stent placement, coronary and other vascular graft procedures (such as tissue rescue following an accident or Thrombotic complications of skin and muscle flap plastic surgery), in diffuse thrombotic/platelet-depleting component conditions such as extensive intravascular coagulation, thrombotic thrombocytopenic purpura, hemolytic syndrome, septicemia Thrombotic complications, adult respiratory distress syndrome, antiphospholipid syndrome, heparin-induced thrombocytopenia and pre-convulsions/convulsions, or venous thrombosis such as deep vein thrombosis of the lower extremities, veno-occlusive disease, hematological conditions such as myeloproliferative disorders, including Thrombocytosis, sickle cell disease; or prevention of mechanically induced in vivo platelet activation, such as cardiopulmonary bypass and extracorporeal membrane oxygenation (prevention of microthrombosis), mechanically induced in vitro platelet activation, such as for preservation of blood products, e.g. , platelet concentrates, or shunt occlusion (such as in renal dialysis and plasmapheresis), vascular injury/inflammation such as vasculitis, arteritis, glomerulonephritis, inflammatory bowel disease, and organ transplant rejection, symptoms (such as partial headache) secondary to thrombosis.
In some embodiments, the antibodies disclosed herein can be administered to patients who are about to receive, or have received, therapy including ticagrelor, and who require or will require therapy for bleeding or potential bleeding associated with coronary artery bypass grafting Surgery (CABG), cardiothoracic surgery, mediastinal reexploration, postoperative stroke, mechanical ventilation, prolonged stay in intensive care unit, non-cardiac surgery (eg, neurological or ophthalmic surgery, renal column surgery, intracranial surgery) , orbital surgery, orthopedic surgery, nephrectomy, partial colectomy, etc.). Thus, the methods provided herein can encompass co-treatment with ticagrelor (simultaneous), or administration of the antibody within a period of time (eg, minutes, hours, or days) following administration of ticagrelor. For example, in some embodiments, the method can include administering the antibody to a patient who has been administered ticagrelor within 10 to 120 minutes of administration of ticagrelor. In some embodiments, the method can include administering the antibody to a patient who has been administered ticagrelor within 1 to 48 hours of administration of ticagrelor. In some embodiments, the antibody is administered to a subject to whom ticagrelor has been administered within a period of time that does not allow for metabolism and elimination of ticagrelor and/or its metabolites from the subject.
In some embodiments, the present invention provides a method for inhibiting the effect of ticagrelor or its active metabolite on (P2Y12
) receptor effect method.
In some embodiments, the present invention provides a P2Y that inhibits the binding of ticagrelor or its active metabolite to a patient12
receptor method.
In some embodiments, the present invention provides a method of activating ADP-induced platelet aggregation in a patient who has been administered ticagrelor.
Antibodies of the invention, such as those exemplified in the Examples, may also be used for diagnostic purposes. For example, one or more target agents (ticagrelor or a metabolite thereof) can be detected in a subject's tissues or cells to determine or screen the circulating amount of ticagrelor in the subject. A diagnostic kit can include one or more antibodies, and a detection system for indicating the reaction of the antibodies with ticagrelor or its metabolites, if any are present.
Thus, the present invention designs antibodies for several uses, including therapeutic, diagnostic, and research uses. Diagnostic and research uses can be in vivo or in vitro. set
Another aspect of the present invention is a kit. In one aspect, a kit includes any of the nucleic acid, antibody, expression vector, or host cell compositions or pharmaceutical compositions described above, along with instructions or labeling to guide proper use or administration. Optionally, the kit also includes one or more containers and/or syringes or other devices to facilitate delivery or use. All or any subset of components designed for use in conducting research assays, diagnostic assays, and/or for administering therapeutically effective amounts of the present invention are packaged in a kit. Similarly, the kit can include instructions for preparing the antibody by, for example, culturing, under suitable conditions, a host cell expressing a nucleic acid encoding an antibody of the invention. By way of additional example, a kit for therapeutic administration of an antibody of the invention can include a solution of a pharmaceutical formulation containing the antibody, or a lyophilized formulation of the antibody, and the composition is administered to a person in need thereof Instructions for the patient and/or for rehydration of the lyophilized product. In certain embodiments, the kit further comprises ticagrelor (eg, BRILINTA™, BRILIQUE™) in a formulation suitable for administration to a subject. In these embodiments, the kit further comprises a formulation for administration of both the antibody and ticagrelor in need of treatment with the antibody, ticagrelor, or both the antibody and ticagrelor Patient description.
The present invention also covers the final packaged and labelled medicinal product. This article of manufacture includes suitable unit dosage forms in suitable containers or contents, such as glass vials or other hermetically sealed containers. In the case of dosage forms suitable for parenteral administration of the active ingredient (eg, the antibody and/or ticagrelor formulations described above), it is sterile and suitable for administration as a particle-free solution. In certain aspects, the formulation is suitable for the injectable route of administration. In some embodiments, the administration is subcutaneous. In some embodiments, the administration is intravenous. Thus, routes of administration including injection or infusion into humans or animals are contemplated.
In a particular aspect, the formulations of the present invention are formulated as sterile liquids in single-dose vials. Exemplary containers include, but are not limited to, vials, bottles, pre-filled syringes, IV bags, blister packs (including one or several pills). As the case may be, associated with this container(s) is a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, the notice reflecting /or approved by the agency for administration.
As with any pharmaceutical product, packaging materials and containers are designed to protect the stability of the product during storage and transportation. In addition, the products of the present invention include instructions for use or other informational material advising a physician, technician or patient on how to appropriately prevent or treat the disease or condition in question. In other words, an article of manufacture includes instructional components that indicate or suggest dosing regimens including, but not limited to, actual dosages, monitoring procedures, etc., and other monitoring information.
Kits for diagnostic assays may include a solution or lyophilized preparation of an antibody containing the antibody of the invention, wherein the antibody specifically binds to ticagrelor and/or its metabolites, and reagents for detecting the antibody. The antibody can be labeled according to methods well known in the art and described herein, including but not limited to, such as small molecule fluorescent tags, proteins such as biotin, GFP or other fluorescent proteins, or epitope sequences such as his or myc to identify. Similarly, a primary antibody for detection of the antibody can be included in the kit. Primary antibodies can be directed to sequences on the antibody or to labels, tags, or epitopes used to identify the antibody. Primary antibodies can then be labeled for detection or if further signal amplification is desired, primary antibodies can be detected by secondary antibodies (which may also be included in the kit).
A kit also designed for research use. Such kits may, for example, be similar to kits intended for diagnostic or therapeutic use but further include a label indicating that the kit and its use are limited to research purposes.Example List of Abbreviations abbreviation explain
ACN Acetonitrile
Br wide
BSA Bovine Serum Albumin
CV column volume
d double
dd double double
DCM Dichloromethane
DMF N,N-Dimethylformamide
DMSO Dimethyl sulfite
DPBS Dubeca's Phosphate Buffered Saline
EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
EtOAc Ethyl acetate
FA Formic acid
HOAc Acetic acid
HPLC High Performance Liquid Chromatography
HRMS High Resolution Mass Spectrometry
HTS High Throughput Screening
HTRF®
Homogeneous Transit Fluorescence
HYFLO®
Filter aid, flux calcined, treated with sodium carbonate
Hz Hertz
J Coupling constant
LC Liquid chromatography
m multiple
MS Mass Spectrometry Analysis
NMR Nuclear Magnetic Resonance
OAc Acetate
Pd/C Palladium on Carbon
pM Pimole
PK/PD Pharmacokinetics/Pharmacodynamics
KF Potassium Fluoride
q Quadruple
r.t. room temperature
s Single weight
sat saturated
scFv Single-chain variable fragment
t triple
TFA Trifluoroacetic acid
TEA Triethylamine
TBME tert-butyl methyl ether
THF Tetrahydrofuran
TIM Ticagrelor inactive metabolite
TLC Thin Layer Chromatography
TR-FRET time-lapse fluorescence resonance energy transferexample 1 : Preparation and characterization of hapten
This example describes methods for synthesizing, optimizing, isolating, and characterizing several haptens for generating exemplary antibodies as described herein. Such haptens include ticagrelor, ticagrelor metabolites (TAM and TIM), biotinylated ticagrelor, and biotinylated adenosine (see, eg, in unbiotinylated form Figure 2) of the chemical hapten structure. Ticagrelor was synthesized as described in International Patent Publication WO 2000/034283 (Guile et al., 2000) and TAM was synthesized as described in International Patent Publication WO 1999/005143 (Guile et al., 1999), each is incorporated herein by reference in its entirety.
Direct phase chromatography was performed using Biotage Silica 40S, 40M, 12i or Merck Silica 60 (0.063 to 0.200 mm). Flash chromatography was performed using standard glass or plastic columns or on a Biotage Horizon system. Chemical shifts are given in ppm using solvent as internal standard. Protons such as N recorded on heteroatoms only when detected in NMRH
and OH
Protons and thus can be ignored.Example 1.1 : Biotinylated ticagrelor
N-(2-(((1S,2S,3S,4R)-4-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)- 5-(Propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,3-dihydroxycyclopentyl)oxy)ethyl) -6-(6-(5-((3aS,4S,6aR)-2-oxyhexahydro-1H-thieno[3,4-d]imidazol 1-4-yl)pentamido)hexyl amide group) hexamethylene amide (1.1
)
(i) Preparation of methanesulfonic acid 2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl) Amino-5-(propylthio-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,2-dimethyltetrahydro-3aH-cyclopentane [d][1,3]dioxol-4-yl)oxy)ethyl ester (1.a
)
At 0 °C, mesylate chloride (0.086 mL, 1.10 mmol) was added dropwise to 2-(((3aR,4S,6R,6aS)-6-(7-((((1R,2S)-2- (3,4-Difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl )-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)oxy)ethanol (see Springthorpe, B. et al. Bioorg.Med Chem. Lett., 2007, 17, 6013-6018) (0.563 g, 1.0 mmol) and TEA (0.209 mL, 1.50 mmol) in DCM (5 mL). The mixture was stirred from 0°C to about 5°C over 3 h. The reaction mixture was diluted with DCM (30 mL) and washed with water (5 mL). The mixture was dried by passing through a phase separator. Evaporation of solvent and co-evaporation from toluene gave the title compound as a thick yellow oil (1.a
) (714 mg, 111%), which was used crude without further purification.1
H NMR (400 MHz, CDCl3
) δ 1.02 (dd, 3H), 1.3 – 1.47 (m, 5H), 1.55 (s, 3H), 1.72 (d, 2H), 2.20 (d, 1H), 2.6 – 2.71 (m, 2H), 2.97 ( s, 3H), 3 – 3.19 (m, 3H), 3.57 – 3.68 (m, 1H), 3.69 – 3.79 (m, 1H), 4.02 (td, 1H), 4.13 – 4.24 (m, 2H), 4.78 ( dd, 1H), 5.13 (td, 1H), 5.57 (s, 1H), 6.50 (s, 1H), 7.03 (s, 1H), 7.07 – 7.16 (m, 2H).19
F NMR (376 MHz, CDCl3
) δ -141.37 (J = 21.3), -138.10 (J = 21.3).
(ii) Preparation of 3-((3aS,4R,6S,6aR)-6-(2-azidoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1, 3] Dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H- [1,2,3]Triazolo[4,5-d]pyrimidin-7-amine (1.b
)
Methanesulfonic acid 2-(((3aR,4S,6R,6aS)-6-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino) -5-(Propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,2-dimethyltetrahydro-3aH-cyclopenta[ d][1,3]dioxol-4-yl)oxy)ethyl ester (1.a) (0.641 g, 1 mmol) and sodium azide (0.070 mL, 2.00 mmol) in DMF ( The mixture in 7 mL) was heated to 60 °C under nitrogen atmosphere for 15.5 h. A white precipitate formed. Water (20 mL) was added and the product was extracted twice with TBME (100+40 mL). Via Na2
SO4
Dry the organic phase. The organic phase was filtered and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on a 2 x 8 cm silica column using heptane/EtOAc 1/1 as eluent (TLC using heptane/EtOAc 1/1 (Rf product=0.5) ). Collection of relevant fractions and evaporation of the solvent gave the title compound as a clear thick oil (1,b
) (514 mg, 87%).1
H NMR (400 MHz, CDCl3
) δ 1.00 (s, 3H), 1.33 – 1.42 (m, 5H), 1.59 (s, 3H), 1.73 (d, 2H), 2.17 (s, 1H), 2.68 (t, 2H), 2.96 – 3.17 ( m, 3H), 3.19 – 3.33 (m, 2H), 3.52 – 3.63 (m, 1H), 3.72 (ddd, 1H), 4.03 (td, 1H), 4.79 (dd, 1H), 5.13 (td, 1H) , 5.54 (dd, 1H), 6.43 (s, 1H), 6.96 – 7.23 (m, 3H).
(iii) Preparation of intermediate 3-((3aS,4R,6S,6aR)-6-(2-aminoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1 ,3]dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H -[1,2,3]Triazolo[4,5-d]pyrimidin-7-amine (1.c
)
3-((3aS,4R,6S,6aR)-6-(2-azidoethoxy)-2,2-dimethyltetrahydro-3aH in EtOH (99.5 %) (2 mL) -Cyclopenta[d][1,3]dioxol-4-yl)-N-((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)-5 -(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-7-amine (1.b
) (62.0 mg, 0.11 mmol) was added to Pd/C (5% Pd, 50 wt% Pd/C, 22.46 mg, 5.28 µmol) and the mixture was hydrogenated at atmospheric pressure for 2 h. The reaction mixture was filtered through HYFLO® and the plug was further rinsed with EtOH (99.5%). The solvent was removed under reduced pressure, the residue was redissolved in DCM (2 x 2 mL) and the solvent was removed under reduced pressure. Flash chromatography on a 2 x 2 cm silica column using DCM/NH3
The residue was purified 95/5 in MeOH (sat.) as eluent. Collect the relevant fractions to obtain the title compound (1.c
) (41 mg, 69%).1
H NMR (400 MHz, CDCl3
) δ 0.98 (m, 3H), 1.28 – 1.46 (m, 7H), 1.54 (s, 3H), 1.62 – 1.81 (m, 2H), 2.15 (s, 1H), 2.48 – 2.81 (m, 4H), 3.07 (tt, 3H), 3.34 – 3.47 (m, 1H), 3.53 (ddd, 1H), 3.99 (td, 1H), 4.79 (dd, 1H), 5.12 (td, 1H), 5.52 (dd, 1H) , 7.02 (s, 1H), 7.09 (dt, 2H), 7.23 (s, 1H).19
F NMR (376 MHz, CDCl3
) δ -141.43 (J
= 21.3), -138.13 (J
= 21.3).
(iv) Preparation of intermediate product (1S,2S,3S,5R)-3-(2-aminoethoxy)-5-(7-(((1R,2S)-2-(3,4-difluoro) Phenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)cyclopentane-1, 2-Diol (1.d
)
A precooled mixture of TFA (8 mL, 103.84 mmol) and water (0.88 mL, 48.85 mmol) at ice/water bath temperature was added to the precooled mixture with 3-((3aS,4R,6S,6aR)-6-( 2-Aminoethoxy)-2,2-dimethyltetrahydro-3aH-cyclopenta[d][1,3]dioxol-4-yl)-N-((1R,2S )-2-(3,4-difluorophenyl)cyclopropyl)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidine-7- amine(1.c
) (340 mg, 0.61 mmol) in a flask. The reaction mixture was stirred at 0 to 5 °C for 1 h. The solvent was removed under reduced pressure and the residue was dissolved in DCM (100 mL) and used NaHCO3
(saturated 10 mL) washed. Brine (5 mL) was added to the aqueous phase and this was extracted with EtOAc (30 mL). Via Na2
SO4
The combined organic phases were dried. Filtration followed by evaporation of the solvent gave the crude product as an off-white solid. by preparative HPLC on an XBridge C18 column (10 μm 250 x 50 ID mm) using a 35 to 75% gradient of ACN in H2
O/ACN/NH3
The compound was purified with buffer in 95/5/0.2 at a flow rate of 100 mL/min for 20 minutes. The compounds were detected by UV at 298 nm. The peak fractions were evaporated to dryness under reduced pressure. The residue was dissolved in DCM and filtered through a phase separator. The solvent was removed under reduced pressure to obtain the title compound (1.d
) (213 mg, 67.5%). LC-MS m/z 522.3 (M+H)+
.
(v) Preparation of compound N-(2-(((1S,2S,3S,4R)-4-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl )amino)-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl)-2,3-dihydroxycyclopentyl)oxy yl)ethyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentane Amino) hexamido) hexamide. (1.1
)
6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentamido)hexanoide Amino)hexanoic acid 2,5-di-oxypyrrolidin-1-yl ester (21.77 mg, 0.04 mmol) was added to (1S,2S,3S,5R)-3-(2-aminoethoxy) -5-(7-(((1R,2S)-2-(3,4-difluorophenyl)cyclopropyl)amino)-5-(propylthio)-3H-[1,2,3 ]triazolo[4,5-d]pyrimidin-3-yl)cyclopentane-1,2-diol (20 mg, 0.04 mmol) in dry DMF (1.0 mL) and the mixture was placed in Under nitrogen atmosphere and stirred at room temperature for 6 h. The solvent was removed under reduced pressure at 40°C. by preparative HPLC on a Kromasil C18 column (10 μm 250 x 20 ID mm) using a 20 to 60% gradient of ACN in H2
The compound was purified with buffer in O/ACN/FA 95/5/0.2 at a flow rate of 19 mL/min for 20 minutes. The compound was detected by UV at 298 nm. Peak fractions were collected, concentrated, and lyophilized to obtain the title compound (1.1
) (21.4 mg, 57.3%).1
H NMR (600 MHz, DMSO): presence of two rotamers (ratio 5:1), signal obtained from major rotamer at δ 0.81 (t, 3H), 1.15 – 1.64 (m, 20H), 2.03 (ddd, 7H), 2.12 (ddd, 1H), 2.57 (d, 1H), 2.59 – 2.67 (m, 1H), 2.77 – 2.89 (m, 2H), 2.93 (dd, 1H), 2.96 – 3.01 ( m, 4H), 3.05 – 3.12 (m, 1H), 3.15 (td, 1H), 3.18 – 3.25 (m, 2H), 3.39 – 3.46 (m, 1H), 3.48 (tt, 1H), 3.7 – 3.76 ( m, 1H), 3.92 (s, 1H), 4.08 – 4.14 (m, 1H), 4.30 (dd, 1H), 4.54 (dd, 1H), 4.95 (q, 1H), 5.06 (s, 1H), 5.13 (d, 1H), 6.35 (s, 1H), 6.42 (s, 1H), 7.07 (d, 1H), 7.31 (ddt, 2H), 7.71 (dt, 2H), 7.82 (t, 1H), 9.36 ( d, 1H). The signal obtained from the lesser rotamer was chosen at δ 0.98 (CH 3
), 8.95 (ArNH
).
[C45
H65
F2
N11
O7
S2
]+
Calculated HRMS: 974.4556; Measured: 974.4585 (M+H)+ example 1.2 : Biotinylated adenosine
N-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl)-6-( 6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentamido)hexanoamido)hexane amide (1.2
)
(i) Preparation of N-(((3aR,4R,6R,6aR)-6-(6-amino-9H-purin-9-yl)-2,2-dimethyltetrahydrofuran[3,4-d] [1,3]Dioxol-4-yl)methyl)-6-(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[ 3,4-d]imidazol-4-yl)pentamido)hexanoamido)hexanoamido (1.e
)
DMF (2 mL) was added to 6-(6-(5-((3aS,4S,6aR)-2-oxyhexahydro-1H-thieno[3,4-d]imidazole at room temperature -4-yl)pentamido)hexanoamido)hexanoic acid 2,5-di-oxypyrrolidin-1-yl ester (55.6 mg, 0.10 mmol) and 9-((3aR,4R,6R, 6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purine-6 - Amine (30 mg, 0.10 mmol) (see Austin, D.J. and Liu, F., Tetrahedr. Lett.,2001
, 3153-3154) and the reaction mixture was placed under nitrogen atmosphere and stirred for 1 h and 45 min to obtain (1.e
). The solvent was then removed under reduced pressure. Used as crude product without further purification. LC-MS m/z 759 (M+H)+
, 757(M-H)-
.
(ii) Preparation of final compound N-(((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl-3,4-dihydroxytetrahydrofuran-2-yl)methyl )-6-(6-(5-((3aS,4S,6aR)-2-oxyhexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentamido)hexane amide group) hexamethylene amide (1.2
)
A mixture of TFA (1.8 ml, 23.36 mmol) and water (0.2 mL, 11.10 mmol) was added to crude N-(((3aR,4R,6R,6aR)-6-(6-amino-9H-purine-9 -yl)-2,2-dimethyltetrahydrofuran[3,4-d][1,3]dioxol-4-yl)methyl)-6-(6-(5-((3aS ,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentamido)hexanoamido)hexanoamido (1.e
) (76 mg, 0.1 mmol) and the reaction mixture was stirred at 0 °C for 1 hour 25 minutes. The solvent was removed under reduced pressure and the residue was dissolved in DMSO. by preparative HPLC on an XBridge C18 column (10 μm 250 x 19 ID mm) using a 5 to 45% gradient of CAN in H2
O/ACN/NH3
The compound was purified with buffer in 95/5/0.2 at a flow rate of 19 mL/min for 20 minutes. The compound was detected by UV at 259 nm. The peak fractions were concentrated and freeze-dried to obtain the title compound as a white fluffy solid (1.2
) (50 mg, 69.6%).1
H NMR (600 MHz, DMSO, 40°C) δ 1.17 – 1.26 (m, 4H), 1.27 – 1.4 (m, 6H), 1.43 – 1.54 (m, 7H), 1.62 (ddt, 1H), 1.99 – 2.06 (m, 4H), 2.12 (t, 2H), 2.58 (d, 1H), 2.82 (dt, 1H), 2.96 – 3.05 (m, 4H), 3.05 – 3.14 (m, 1H), 3.36 (dt, 1H) ), 3.44 (dt, 1H), 3.96 (dd, 1H), 4.04 (dd, 1H), 4.11 – 4.15 (m, 1H), 4.29 – 4.33 (m, 1H), 4.67 (dd, 1H), 5.16 ( d, 1H), 5.38 (d, 1H), 5.84 (d, 1H), 6.29 (s, 1H), 6.33 (d, 1H), 7.25 (s, 2H), 7.64 (dt, 2H), 8.11 (t , 1H), 8.16 (s, 1H), 8.31 (s, 1H). For [C32
H50
N10
O7
S]+
Calculated HRMS: 719.3657; Measured: 719.3667 (M+H)+ example 2 : Isolation and identification of anti-ticagrelor /TAM Antibody
This example illustrates strategies and techniques that can be used to prepare antibodies against ticagrelor and its metabolites, compounds that are structurally similar to ATP and contain adenosine-like cores (Springthorpe et al., 2007 Bioorg Med Chem Lett. 17:6013-6018 ). The chemical structures of ticagrelor, ticagrelor active metabolite (TAM) and ticagrelor inactive metabolite (TIM) are inpicture 2
displayed in. As discussed above, the antibodies disclosed and generated herein can bind and neutralize ticagrelor and TAM and can bind to TIM, but do not bind or significantly inhibit other structurally related compounds such as adenosine. While binding activity to TIM is an optional feature of the antibodies disclosed herein, since TIM typically represents a small or insignificant fraction of ticagrelor metabolites, antibodies exhibiting binding activity to TIM are not expected to affect the desired Antidote dose.
Targeting commonly used epitopes (ie, the unique R groups of ticagrelor and TAM (difluorophenyl-cyclopropyl and thiopropyl substituents)) to confer antibody binding specificity and selectivity to those compounds . The epitope of interest ispicture 2
is delineated by a dashed line. The haptens described in Example 1 were used to direct antibody epitopes towards difluorophenyl-cyclopropyl and thiopropyl substituents. As described in Example 1, the linkers for the biotinylated haptens (biotinylated ticagrelor and biotinylated adenosine) are located on the diol groups. This strategy allows the preparation of antibodies with binding specificity to unmodified di-fluorophenyl-cyclopropyl and thiopropyl, biotinylated ticagrelor/TAM, and can also be used to screen and deselect for binding to adenocarcinoma Antibody library of glycosides.
Using known techniques, human scFv phage display libraries can be used to generate scFv antibodies, and specific scFvs are isolated from the library in a series of cycles of repeated selection for biotinylated ticagrelor and de-selection of biotinylated adenosine, essentially The above is as described in Lloyd et al., 2009 PEDS 22:159-168 (incorporated herein by reference). Several individual clones were selected from the selection output of cycle 2 and cycle 3, and the scFv was expressed in the bacterial periplasm and screened for specificity in three parallel biochemical assays. This assay screens for i) binding to biotinylated ticagrelor (assay 1), ii) binding to biotinylated adenosine (assay 2) and iii) in the presence of a 50-fold excess of unmodified ticagrelor Binding to biotinylated ticagrelor (Assay 3) to confirm specificity for ticagrelor but not the biotinylated linker.
Analyses 1, 2 and 3 were performed using the same general techniques and strategies. Briefly, HTS bound to crude periplasmic scFv samples of biotinylated ticagrelor or biotinylated adenosine was performed using HTRF®
Analytical techniques are performed. HTRF®
(Homogeneous Transit Fluorescence) is based on the principle of TR-FRET (Transit Fluorescence Resonance Energy Transfer). Briefly, TR-FRET employs a transition from a donor fluorophore (in this case a europium cryptate) to an acceptor fluorophore (in this case XL665
) energy transfer. The condition is that the donor and acceptor fluorophores are in close enough proximity (approximately <10 nm) that excitation of the europium cryptate donor (337 nm) results in energy transfer to XL665
receptor, which in turn emits a fluorescent signal at 665 nm. This technique can be used to sensitively measure biomolecular interactions by attaching donor and acceptor fluorophores (directly or indirectly) to each binding partner under a specific interaction. Binding of scFv to the HTS form of biotinylated ticagrelor (Assay 1) is shown below and relies on the presence of a chemical tag in both biotinylated ticagrelor and his-tagged periplasmic scFv:
europium cryptate streptavidin: biotinylated ticagrelor: scFv-His: anti-His-XL665
Black shallow well 384-well assay plates were used in an assay volume of 10 μl in buffer including DPBS pH 7.4 (Gibco 14190-086), KF (VWR 103444T) (0.4M) and Tween 20 (Sigma P9416) (0.05%) (Corning/Costar 3676) for this analysis. by adding 5 μl biotinylated ticagrelor (60 nM to obtain 30 nM final concentration), 2 μl of periplasmic scFv sample (20% final concentration) and 3 μl containing europium cryptate-labeled streptavidin Biotin (CisBio 610SAKLB) (4.2 nM to obtain a final concentration of 1.26 nM) and XL665
The assay was established with a solution of labeled anti-His antibody (CisBio 61HISXLB) (40 nM to obtain a final concentration of 12 nM). Negative binding control wells containing all of the above assay components were established except where 2 μl of assay buffer was added in place of the periplasmic scFv. The assay plates were incubated at room temperature for 4 hours and then read on an Envision plate reader using a standard HTRF reading protocol in which samples were excited at 337 nm and differential fluorescence emission was measured at 620 nm and 665 nm.
Raw 665 nm and 620 nm counts were first converted to 665 nm/620 nm ratios and the results were then expressed as ΔF (%) values. Calculate ΔF according to the following equation:
ΔF(%)={((Sample 665/620 ratio)-(Negative 665/620 ratio))/(Negative 665/620 ratio)}x100
(using the negative ratio from negative binding control wells). A scFv that provided a ΔF value greater than 100% in this analysis was defined as a hit.
The same protocol as described above was used to perform crude periplasmic scFv samples against HTS bound to biotinylated adenosine (final assay concentration 30 nM). The form of Analysis 2 can be described as follows:
europium cryptate streptavidin: biotinylated adenosine: scFv-His: anti-His-XL665
Assay 3 employed the same protocol as described above to identify HTS of crude periplasmic scFv samples showing reduced binding to biotinylated ticagrelor in the presence of excess free unmodified ticagrelor. This protocol was modified from Assay 1 by performing Assay 3 in the presence of a 50-fold molar excess of free unmodified ticagrelor (1500 nM).
Hits were defined as binding to biotinylated ticagrelor (ΔF > 100% in Assay 1) but not to biotinylated adenosine (ΔF < 25% in Assay 2) and in excess free The presence of modified ticagrelor reduced binding to biotinylated ticagrelor by >50%. An example of the correlation of analysis 1 and 3 data is inpicture 3
displayed in. Several scFvs showed limited inhibition in the presence of excess free unmodified ticagrelor, suggesting binding interactions with ticagrelor and certain components of the linker. A subset of scFvs were identified that inhibited (>50%) binding to biotinylated ticagrelor in the presence of excess free unmodified ticagrelor. scFvs were classified based on the % inhibition of binding observed in Assays 3 and 1 (50 to 80%, 80 to 90%, >90%), with scFvs providing >90% inhibition (including TICA0072) being preferred for further characterization . Sequence unique scFv hits were converted to Fab and expressed in CHO cells using standard techniques.Fab Expression and Purification
Separate HC and LC expression plasmids were used for transient transfection, based on the expression vector described by Persic et al. The vector was modified to contain the EBV origin of replication (OriP
). The Fab(HC) vector contained only constant region 1 (CH1) and hinge region, CH2 and CH3 were removed. HC and LC DNA were added to 150 mM NaCl and 25-kDa linear PEI (Polysciences Europe, Germany 23966) according to the manufacturer's recommendations. The DNA-PEI complex was then added to Chinese hamster ovary wild-type (CHO wt) cells (Daramola O et al. 2014) suitable for suspension culture derived from the CHOK1 cell line (ECACC number: 85051005). After 7 days, the cells were harvested by centrifugation and the supernatant was filtered. The cell culture supernatant containing the Fab protein was loaded directly into a chromatography column packed with 5 ml CaptureSelect IgG-CH1 (Life Technologies, Carlsbad, USA) using an Äkta purifier (GE Healthcare) at a flow rate of 5 ml/min . The column was equilibrated and washed with phosphate buffered saline (PBS) pH 7.2 and eluted with 20 mM sodium citrate, 150 mM sodium chloride, pH 3.5 (CaptureSelect IgG-CH1) according to the resin manufacturer's instructions . The eluted Fab was adjusted to pH 5.5 and filtered (0.22 μm Steriflip, Millipore EMD, Bethdesa, USA) prior to analysis. Protein concentrations were determined by absorbance at 280 nm using a DU520 UV/vis spectrometer (Beckman Coulter, Brea, USA). Sample purity was determined using a TSK gel G3000SWxl column (Tosoh Bioscience, Tokyo, Japan) and a 1100 HPLC system (Agilent Technologies, Santa Clara, USA) running at 1.0 ml/min.example 3 : Anti-ticagrelor /TAM Of Fab
Structural databases containing commercially available drugs ("DrugsDB", Oprea T.I et al., 2011 Mol. Inform. 30(2-3), 100-111, incorporated herein by reference) were interrogated to determine the presence of ticagrelor Molecules with certain structural similarities. Once determined, these structurally similar molecules were used to test the binding specificity of Fabs in addition to adenosine and its phosphorylated forms (eg, ADP and ATP). The database was queried for molecules with 2D fingerprint similarity, 3D shape, and electrostatic similarity to ticagrelor, based on ticagrelor X-ray and NMR structures. From this computer analysis, a group of 12 compounds including one of six potential synergistic drugs was selected. The structures of these compounds are inpicture 4
displayed in.
Specificity is studied in a competitive binding assay format, in which each test compound is tested for its ability or otherwise to competitively inhibit the interaction of biotinylated ticagrelor with the relevant Fab. HTRF®
A competition assay format was used as listed below, where the objective was to measure competition for biotinylated ticagrelor binding to each His-Fab by a panel of test compounds:
Europium Cryptate Anti-His Antibody: Test His-Fab: Biotinylated Ticagrelor: XL665
Marked streptavidin.
This basic assay format was used to evaluate the selectivity profile of the lead Fab obtained from lead isolation and at the end of the lead optimization period and to generate Fab using the His-Fab expression vector for the purposes of these studies.
In a black shallow well 384-well assay plate (Corning/Costar 3676) in a 20 μl assay volume in a 20 μl assay volume containing DPBS pH 7.4 (Gibco 14190-086), KF (VWR 103444T) (0.4M) and BSA (PAA K05-013) ( 0.1%) buffer for the analysis. Titration involving the addition of 5 μl of biotinylated ticagrelor, 5 μl of each test-selective compound, 5 μl of the relevant His-Fab, and 5 μl of an anti-His antibody (CisBio 61HISKLB) containing the Europium cryptate label (5.33 nM) to obtain a final concentration of 1.33 nM) and XL665
The assay was established with a combined solution of labeled streptavidin (CisBio 611SAXLB) (40 nM to obtain a final concentration of 10 nM). A total binding control well was established containing all of the above assay components except that 5 μl of assay buffer was added in place of the test selective compound. Negative binding control wells were established containing all assay components included in the total binding control wells except that 5 μl of Assay Buffer was added in place of the addition of His-Fab. Depending on the particular experiment, test compound series were titers of 1/2 or 1/3 and optimized for the highest final assay compound concentration on a compound-specific basis. In independent experiments, the concentrations of biotinylated ticagrelor and His-Fab were optimized on the basis of Fab specificity. For the four Fabs studied at the end of the lead isolation period (TICA0010, TICA0049, TICA0053 and TICA0072), the final assay reagent concentrations used are listed in Table 1 below:
Table 1 Antibody ID [Antibody] (nM) [Biotinylated ticagrelor] (nM)
TICA0039 16.0 139.9
TICA0049 16.0 37.9
TICA0053 16.0 70.7
TICA0072 8.0 17.6
For the two Fabs studied at the end of the lead optimization period (TICA0162 and TICA0212), the final assay reagent concentrations used in both cases were 5 nM biotinylated ticagrelor and 1 nM His Fab. Several of the test-selective compounds used in these experiments were dissolved in 100% DMSO and vehicle-related reductions in analytical signal could begin to appear at concentrations greater than about 1% DMSO. To enable subsequent normalization of the data to correct for any such vehicle-related effects, parallel titrations of DMSO alone were included in most experiments, with the final analytical DMSO concentrations reflecting the concentrations of serial dilutions of their test compounds. At the end of the setup procedure, the assay was incubated for 3 hours at room temperature before being read on an Envision plate reader using a standard reading protocol.
For subsequent data analysis, the raw 665 nm and 620 nM counts were first converted to 665 nm/620 nm ratios, which were then used to calculate ΔF% values according to the equation set forth in Analysis 1. Negative ratios used to calculate ΔF% were derived from negative binding control wells. The ΔF value % is then used to calculate the % specific binding value according to the following equation:
Specific binding (%)={(sample ΔF-negative binding ΔF)/(total binding ΔF-negative binding ΔF)}x100
For standard calculations of % specific binding, total binding ΔF was obtained from total binding control wells containing all of the above mentioned components of the assay but without any competing test compound.
In those experiments in which DMSO normalization was applied, the DMSO-normalized % specific binding was calculated essentially according to the equation above, except in this case the total binding ΔF was obtained from fractions containing all of the total binding control wells as well as DMSO Wells whose concentration is equal to their concentration in the relevant sample well.
Results obtained from initial experiments of this type are summarized in Table 2. In three of the four initial Fabs studied (TICA0010, TICA0049 and TICA0053), the compound cangrelor showed competitive inhibition of Fab binding to biotinylated ticagrelor. In the case of TICA0049, both pantoprazole and linezolid showed partial competitive inhibition. As shown in Table 2, TICA0072 Fab showed no inhibition of eleven of the twelve compounds, while pantoprazole showed weak partial inhibition.
Inhibition of unmodified ticagrelor and TAM was observed in all four Fabs tested in this first series of experiments, IC50
Values fall in the 0.1 μM to 0.5 μM range. Competitive inhibition of TIM by two of the four Fabs (TICA0049 and TICA0072) was also tested, however, IC50
Values >50 μM, indicating a greatly reduced affinity for TIM compared to unmodified ticagrelor and TAM. Based on the results in Table 2, TICA0072 was determined to have the most favorable selectivity profile. TICA0049 was identified as a potential spare based on the criterion that this Fab showed minimal binding to cangrelor among the remaining three Fabs.
Table 2. Relative IC for each of the 12 test compounds (FIG. 4) and unmodified ticagrelor, TAM and TIM for inhibition of biotinylated ticagrelor binding to each tested Fab50
value. IC 50 (uM) of various compounds for lead his-Fab
compound TICA0010 TICA0049 TICA0053 TICA0072
fenofibrate NI NI NI NI
Nilvadipine NI NI NI NI
Cilostazol NI NI NI NI
Brady New NI NI NI NI
Regardson NI NI NI NI
cyclothiazide NI NI NI NI
Sai Funing NI NI NI NI
lovastatin NI NI NI NI
Linezolid NI 467.1 NI NI
Simvastatin NI NI NI NI
Cangrelo 102.4 207.9 17.5 NI
pantoprazole NI 263.7 NI 498.0
ticagrelor 0.122 0.257 0.109 0.113
TAM 0.124 0.412 0.134 0.299
TIM NI 53.4 NI 54.4
The NI line is not inhibited.
Further selectivity data for the TICA0049 and TICA0072 Fabs were generated in a second series of experiments, which were re-tested in conjunction with ticagrelor, TAM and TIM and several adenosine related compounds inpicture 4
Four of a group of twelve compounds listed in. Here, a refinement of the earlier study in Table 2, the experiments were designed so that the percent specific binding values were normalized to correct for any non-specific vehicle related effects due to DMSO. An example depicting the data obtained from this second series of experiments is inpicture 5
shown in, together with the ICs tabulated in Table 350
value.
Table 3. In competitive binding selectivity studiespicture 4
Example ICs for subgroup four of the twelve compounds listed in Ticagrelor, TAM, TIM and several adenosine family of compounds50
Results (DMSO normalized) compound TICA0049 TICA0072
Adenosine NI NI
ADP NI NI
2MeS ADP NI NI
ATP NI NI
2 MeS ATP NI NI
Brady New 864.9 NI
Linezolid 902.0 NI
Cangrelo 188.4 NI
pantoprazole 243.5 1546.0
ticagrelor 0.368 0.356
TAM 0.366 0.483
TIM 74.8 119.5
As with the earlier experiments, TICA0072 showed the most favorable selectivity profile, while only pantoprazole showed weak partial inhibition (>1500 μM) of the four compounds tested. In the case of TICA0049, significant inhibition was observed with cangrelor and pantoprazole and weak inhibition was observed with linezolid and bradysin. It should be noted that the absolute IC between the first and second series of experiments for some of the compounds analyzed50
Minor differences in values do not substantially alter the overall conclusion. These differences may arise from a combination of facts: the fact that DMSO normalization was consolidated into the second series of experiments, and the fact that in several cases we attempted to measure very weak inhibition.
IC measured for ticagrelor and TAM for both TICA0049 and TICA007250
values range from 0.3 μM to 0.5 μM, while IC for TIM50
Values were greater than 2 log values, above 74.8 μM and 119.5 μM, respectively. For both TICA0049 and TICA0072 Fab, slight inhibition was observed in adenosine, ADP, ATP and the methylthio derivatives of the latter two compounds, however this was only detected at the highest compound concentration tested and was not considered Remarkably.
It was concluded from this that TICA0072 was the only Fab deemed specific for ticagrelor and TAM.example 4 : Anti-ticagrelor /TAM Fab Affinity measurement
The affinity of the anti-ticagrelor Fabs generated above was determined on Octet Red384 using a biolayer interferometer. For affinity measurements, anti-ticagrelor Fab antibodies were diluted in assay buffer (PBS, Tween20 0.05%, BSA 0.02%) to a concentration of 2x the final assay concentration, eg 200 nM. 10 point 2-fold serial dilutions of ticagrelor were prepared in Greiner polypropylene 96-well plates. Equal volumes (eg, 70 μL plus 70 μL) of diluted antibody and free ticagrelor were then transferred to a second Greiner polypropylene plate. The samples were mixed by pipette, covered with a plate lid and allowed to equilibrate at room temperature for 3 to 5 days. After equilibration, 60 μL of antibody/ticagrelor titers were transferred in duplicate to a 384-well black sloped-bottom polypropylene plate. Biotinylated ticagrelor was diluted to 250 nM in assay buffer and added to alternating wells of 2 columns prior to addition to a 384-well assay plate, the remaining wells contained assay buffer only. Pre-soak the streptavidin biosensor in assay buffer for at least 10 minutes. The sample plate and biosensor were then placed on the stage of the OctetRed384.
All analyses were performed at room temperature. After baseline equilibration in assay buffer for 60 sec, biotinylated ticagrelor was loaded onto the streptavidin biosensor for 300 sec, followed by 600 sec in assay buffer to establish a new baseline. The antibody/ticagrelor mixture was then allowed to associate with the biotinylated ticagrelor sensor surface for 30 to 600 sec, depending on the concentration of antibody used. The resulting association period data were analyzed using OctetRed data analysis software. For each sample, the signal was aligned to the baseline and the reference sensor signal was subtracted (no antibody control), and the data was then exported for analysis using KinExA n-type curve analysis software. Equilibrium KD of anti-ticagrelor Fab antibodies was determined using Constant Partner assay. The data show that Fabs TICA0072 and TICA0049 have ticagrelor affinity of 7.4 nM and 11.6 nM, respectively (Table 4).
Table 4. Equilibrium affinity analysis of anti-ticagrelor Fabs Antibody ID hapten Balance KD 95% confidence interval
TICA0072 ticagrelor 7.43 nM 1.75-21.46 nM
TICA0049 ticagrelor 11.6 nM 1.7-66.5 nM
example 5 : Optimized anti-ticagrelor /TAM Antibody TICA0072
Antibody TICA0072 was optimized using affinity-based phage selection. Variable heavy (VH) complementarity determining region (CDR) 1, 2 or 3 or variable light (VL) chain CDR 1, 2 or 3 using standard molecular biology techniques as described (Clackson and Lowman 2004 Practical Approach Series 266). The oligonucleotide-directed mutagenesis creates a large scFv library derived from the lead scFv sequence. The library was subjected to affinity-based phage display selection to select for variants with higher affinity for ticagrelor and TAM. Briefly, scFv phage particles were grown in solution with reduced biotinylated ticagrelor concentrations (a typical example would be 20 nM to 20 pM after four selection cycles), essentially as previously described (Thompson et al. Human, 1996 J Mol Biol. 256(1):77-88). Preparation of crude scFv-containing periplasmic extracts obtained from a representative number of individual scFv outputs from CDR targeting selection and in HTRF®
Screening in an epitope competition assay format designed to screen for improvement in affinity relative to TICA0072.
Briefly, to screen for scFv and Fab variants with improved affinity, HTRF®
Epitope competition analysis was performed by testing scFv variants based on competition between the parental TICA0072 IgG and biotinylated ticagrelor. This assay was used as a primary single-site HTS to screen crude periplasmic extract scFv samples and as a multi-site secondary profiling assay to measure the IC of purified scFv and Fab variants against the parental TICA007250
Improvement in value. Although epitope competition assays such as the one described herein are not generally used to determine absolute affinity values, such assays can be used as the basis for affinity-based HTS. In addition, the IC of purified scFv/Fab variants (relative to the parental scFv/Fab)50
Fold improvement can provide a good indication of overall fold gain in affinity and can represent an efficient way to affinity fractionate scFv/Fab variants in lead optimization activities. The format of the TICA0072 parental IgG-based epitope competition assay described herein is listed below:
Europium-labeled streptavidin: biotinylated ticagrelor; TICA0072 IgG: XL665
Labeled anti-human Fc antibody
Black shallow well 384-well assay plates (Corning/Costar 3676) in buffers including DPBS pH 7.4 (Gibco 14190-086), KF (VWR 103444T) (0.4M) and Tween 20 (Sigma P9416) (0.05%) for this analysis. For single-site testing of crude periplasmic scFv variants, a 10 μl assay volume was used, however when the multi-site secondary IC50
When testing purified scFvs and Fabs in profiling assays, a 20 μl assay volume was used.
For single point HTS, by adding 3 μl TICA0072 IgG (53.3 nM to obtain 16 nM final concentration), 2 μl crude periplasmic extract scFv sample, 2.5 μl biotinylated ticagrelor (8 nM to obtain 2 nM final concentration) and 2.5 μl containing Europium-labeled streptavidin (CisBio 610SAKLB) (3 nM for final assay concentration of 0.75 nM) and XL665
The assay was set up with a combined solution of the labeled anti-human Fc antibody (CisBio 61HFCXLB) (30 nM to obtain a final assay concentration of 7.5 nM). The parental TICA0072 crude periplasmic scFv was used as a reference point and the HTS was configured to identify variants that provided improved inhibition relative to the parent. Total binding control wells contained all assay components except for the addition of 2 μl assay buffer in place of the scFv sample. Negative binding control wells contained all of the total binding control well components except for the addition of 3 μl assay buffer in place of TICA0072 IgG.
Multipoint IC for purified scFv/Fab variants50
For testing, scFv or Fab variants were tested by adding 5 μl TICA0072 IgG (53.3 nM to obtain a final concentration of 16 nM), 5 μl 1/3 titrated purified test scFv or Fab variant, 5 μl biotinylated ticagrelor (8 nM to obtain a final concentration of 2 nM (scFv profiling), 4 nM to obtain a final assay concentration of 1 nM (Fab profiling), and 5 μl of streptavidin (CisBio 610SAKLB) containing Europium (3 nM, for 0.75 nM final assay concentration) and XL665
The assay was set up with a combined solution of the labeled anti-human Fc antibody (CisBio 61HFCXLB) (30 nM to obtain a final assay concentration of 7.5 nM). The purified parental TICA0072 (scFv or Fab) was used as a benchmark in all experiments to allow the IC measured using the optimized variant (scFv or Fab)50
The improvement can be expressed as a fold improvement relative to the parental TICA0072. Total binding control wells contained all assay components except where 5 μl of assay buffer was added in place of purified scFv or Fab samples. Negative binding control wells contained all components of total binding control wells except for the addition of 5 μl assay buffer in place of TICA0072 IgG.
Single-point HTS and multi-point IC under analysis50
In the spectroscopic version, plates were incubated for 3 hours at room temperature and then read on an Envision plate reader using a standard HTRF reading protocol in which samples were excited at 337 nm and time-lapse fluorescence emission was measured at 620 nm and 665 nm.
Raw 665 nm and 620 nm counts were used to calculate ΔF (%) and % specific binding according to the equations described earlier in Assays 1 and 4, respectively. For multipoint secondary spectroscopy experiments, IC was determined by Graphpad Prism software using a sigmoid dose-response (variable slope) curve fit (4-parameter logistic equation)50
value.
Hits identified in the screening, ie scFv variants showing significantly improved inhibitory effects when compared to the parental TICA0072 scFv, undergo DNA sequencing, and will subsequently be obtained from the variable heavy CDR1, CDR2 or CDR3 and variable light libraries Unique variants exported by CDR1, CDR2 or CDR3 were prepared as purified scFvs and re-tested in the same assay to determine concentration-response IC50
curve. The most improved ICs will then be displayed50
Valued scFv variants were prepared as Fab and tested in the second generation epitope competition assay described below.for filtering / Class highest affinity Fab The second generation epitope competition analysis
For efficient differentiation between very high affinity purified Fabs at the end of the lead optimization campaign, another HTRF was implemented®
An epitope competition assay, however in this case the assay was based on the competitive inhibition of the intermediate affinity-optimized TICA0072 lineage IgG (TICA0159) binding to biotinylated ticagrelor rather than the parental TICA0072 IgG. This analysis is for multipoint ICs only50
Spectralized format (rather than HTS format) and substantially the same as the multipoint IC for the purified Fab variant in the parental TICA0072 IgG-based epitope competition assay in Assay 5 (above)50
The methods given for spectrography were performed in the same way. The only difference was the use of TICS0159 IgG instead of TICS0072 IgG at the appropriate point in the assay establishment but the same final assay concentration of 16 nM. In all other aspects, this analysis was performed exactly as described above in the multipoint secondary spectrometry version for purified Fab.
The second generation assay using the partially optimized antibody TICA0159 instead of TICA0072 was able to more efficiently differentiate and rank the highest affinity Fabs than could be achieved by a TICA0072-based epitope competition assay.
The most improved VH identified was the CDR3 variant TICA0162. The most improved VL was identified as the CDR3 variant TICA0152. To generate further affinity improvements, the different CDRs derived from the improved antibodies were combined into new Fabs using standard molecular biology techniques. From this recombination procedure, the combination of TICA0162 and TICS0152 resulted in a new Fab TICA0212 with a further improved epitope competition profile. The TICA0072, TICA0152, TICA0162 and TICA0212 Fab competition curves depicted in the second generation epitope competition assay are inpicture 6
shown in , the measured IC50
The values are in Table 5. TICA0212 Display IC50
about a 2 log improvement relative to the parental TICA0072 Fab.
Table 5: ICs of the optimized antibody Grelor Fab listed in the second generation epitope competition assay50
data. Fab IC50 (nM) improvement factor
TICA0072 1714.0 0
TICA0152 73.5 23.3
TICA0162 16.3 105.2
TICA0212 12.0 142.8
Example 6 : Optimized Anti-Ticagrelor /TAM Fab Affinity measurement
The affinity of the anti-ticagrelor/TAM Fab generated in Example 5 was determined using KinExA3200. For the KinExA affinity measurement, first by combining it with 1 mg streptavidin in 50 mM NaHCO3
Beads (600 mg azlactone beads) were prepared in the reaction overnight. After blocking with 2 variations of Tris buffer (1M Tris pH 8.7, 10 mg/mL BSA), the streptavidin-coated beads were resuspended in a total volume of 8 mL. Beads were washed thoroughly with PBS (1.33 mL, equal to 100 mg of initially dry azlactone beads), then allowed to bind to approximately 2.5 μg biotin-ticagrelor in 1 mL of PBS for 10 minutes with occasional agitation. The resulting biotin-ticagrelor coated beads were washed with PBS and then resuspended in 0.1% BSA and 0.02% NaH3
in 50 mL of PBS and stored at room temperature until transfer to a KinExA bead vial.
Antibody/ticagrelor sample preparation was performed essentially as previously described. In assay buffer (PBS, Tween20 0.05%, BSA 0.02%, 0.02% NaN3
), the anti-ticagrelor Fab antibody was diluted to a concentration of 2-fold the final assay concentration (eg, 200 nM). Prepare 10 point 2-fold serial dilutions of ticagrelor in Falcon 50 mL polypropylene tubes. Equal volumes (eg, 5 mL plus 5 Ml) of diluted antibody and free ticagrelor were then transferred to a second Falcon polypropylene tube. Samples were mixed by pipette and allowed to equilibrate at room temperature for 3 to 5 days. After equilibration, the sample tube was transferred to the KinExA 3200 for analysis.
All analyses were performed at room temperature. The antibody/ticagrelor mixture was sampled and allowed to mix with biotin-ticagrelor beads while washing away unbound free ticagrelor. Bound antibodies were then detected using DyLight649 labeled mouse anti-human heavy and light chain antibodies. Sample volume (300 to 1300 μL) and injection time (90 to 120 s) varied with concentration and 2 to 3 beads per sample. Data were analyzed using KinExA n-type curve analysis software. Equilibrium KD of anti-ticagrelor Fab antibodies was determined using Constant Partner assay.
As in the previous example, the Fab was allowed to equilibrate at room temperature in the presence of a 20-fold excess concentration of unmodified ticagrelor or TAM. Biotinylated ticagrelor was loaded on the surface of streptavidin-coated beads. After equilibration, remaining free antibody was allowed to bind to biotinylated ticagrelor. Bound antibodies were then detected using DyLight649 labeled mouse anti-human heavy and light chain detection antibodies. Titration of free ticagrelor or TAM was prepared using at least three different pooled concentrations of antibody to generate independent titration curves with at least a 10-fold shift in apparent KD. Data were analyzed using KinExA Pro n-curve analysis software to determine the equilibrium KD of free ticagrelor or TAM. Fab TICA0212 had an affinity of about 20 pM for unmodified ticagrelor and TAM (Table 6). Based on the equilibrium data, TICA0212 demonstrated fairly high affinity (~20 pM) binding to ticagrelor and TAM.
Table 6: Equilibrium affinity analysis of anti-ticagrelor/TAM Fab Antibody ID CDR3 sequence (SEQ ID NO) hapten Balance KD 95% CI
TICA0072 VH GSHLY 99 DFW 100b SASHPPNDALAI (35) VL GTW 91 D 92 I 93 S 94 LSAGL (40) ticagrelor 7.4 nM 1.8-21.5 nM
TICA0152 VH GSHLYDFWSASHPPNDALAI (65) VL GTW LYDRAV GL (70) ticagrelor 43.17 pM 2.8-119.2 pM
TICA0162 VH GS FD Y Y FWSASHPPNDALAI (55) VL GTWDISLSAGL (60) ticagrelor 162.48 pM 125.4-206.3 pM
TICA0212 (MEDI2452) VH GS FD Y Y FWSASHPPNDALAI (75) VL GTW LYDRAV GL (80) ticagrelor 19.6 pM 13.0-28.7 pM
TAM 19.7 pM 4.9-44.7 pM
TIM ~ 20 nM
bold
Variations of sequence residues from the parental TICA0072, and kabat numbers were determined for specific residues in TICA0072.example 7 : Anti-ticagrelor /TAM Fab TICA0162 and TICA0212 specificity
Fabs TICA0162 and TICA0212 were tested for specificity and normalized to DMSO as in Example 3. A summary of all available selectivity data for TICA0162 and TICA0212 is included in Table 7. In addition, plots obtained from the tests obtained in conjunction with ticagrelor, TAM, TIM, and several of the adenosine family of compounds inpicture 4
Examples of data from experiments with five of the twelve compounds listed inpicture 7
displayed in.
Table 7: Relative ICs of twelve structurally related compounds other than ticagrelor, TAM, TIM and adenosine family compounds for inhibiting the binding of biotinylated ticagrelor to TICA0162 and TICA0212 Fab50
value. IC 50 (uM) of various compounds for lead his-Fab
compound TICA0162 TICA0212
fenofibrate NI NI
Nilvadipine NI NI
Cilostazol NI(n=2) NI(n=2)
Brady New NI NI
Regardson NI(n=2) NI(n=2)
cyclothiazide >1000(n=2) NI(n=2)
Sai Funing NI NI
lovastatin NI NI
Linezolid NI NI
Simvastatin NI(n=2) NI(n=2)
Cangrelo NI NI
pantoprazole >1000(n=2) NI(n=2)
Adenosine NI NI
ADP NI NI
2MeS-APD NI NI
ATP NI NI
2MeS-ATP NI NI
ticagrelor 0.023(n=2) 0.035(n=2)
TAM 0.032(n=2) 0.031(n=2)
TIM 19.8(n=2) 28.8(n=2)
The NI line is not inhibited.
As illustrated by the data, TICA0212 (NEDI2452) has substantially comparable binding specificity for ticagrelor and TAM, and binds weakly to TIM. Furthermore, MEDI2452/TICA0212 did not show significant binding to any other structurally related drugs or adenosine related compounds.example 8 : Protein Crystallography
TICA0072 with a C-terminal his-tag was concentrated to 9 mg/ml in PBS. Complex formation was achieved by adding 1 mM ticagrelor in DMSO. The complexes were incubated at room temperature for 2 hours prior to crystallization experiments using the sinking vapor diffusion method. Extensive screening was performed using 3 commercial sieves and several hits were obtained. by Morpheus®
(Molecular Dimensions, UK) hit object grid optimization to obtain the best diffraction crystal. By mixing equal volumes of TICA0072-ticagrelor complex with 12.8% PEG 3350, 12.8% PEG 1000, 12.8% MPD, 1.7% 1,6-hexanediol, 1.7% 1-butanol, 1.7% 1,2 - Propylene glycol, 1.7% 2-propanol, 1.7% 1,4-butanediol, 1.7% 1,3-propanediol, 25 mM imidazole, 25 mM sodium cacodylate, 25 mM MES, and 25 mM Bis-Tris pH 6.5 The crystals for structure determination were grown from the storage tank solution at 20°C. The crystals were flash frozen in liquid nitrogen without any cryoprotectant added.
TICA0212/MEDI2452 in PBS at a concentration of about 15 mg/ml was mixed with ticagrelor to a concentration of 1 mM and incubated for 2 hours at room temperature prior to extensive screening. No spontaneous hits were obtained. Seeds obtained from TICA0072-Ticagrelor crystals were prepared by crushing several crystals in 30 μl well solution and used for MMS (Microseed Matrix Screening) to a broad commercial sieve. Several hits were obtained, but crystals used for structure determination were grown at 20°C in 20% glycerol, 20% PEG 4000, 10% 2-propanol, 0.1 M NaCl and 0.5 M NaAcetate pH 4.6. The drops were prepared using 0.2 μl protein, 0.18 μl well solution and 0.02 μl seed stock solution. The crystals were flash frozen in liquid nitrogen without any cryoprotectant added.
Data were collected at beamline ID23-1 of the European Synchrotron Radiation Facility (Grenoble, France). This data was processed, scaled and further reduced using the AutoProc workflow [Vonrhein, C. et al., Acta Cryst. 2011; D67: 293-302], see Table 8 for statistics. For TICA0072, the initial phase was completed by molecular substitution using the high-resolution Fab structure (PDB identifier 1aqk, [Faber C. et al., Immunotechnology. 1998;3:253-70]) as the starting model. For TICA0212/MEDI2452, the TICA0072 structure was used as the starting model. Model reconstruction using Coot [Bricogne G. et al, (2011). BUSTER version 2.11.4. Cambridge, United Kingdom: Global Phasing Ltd] and use of an automatic pulverizer [Emsley, P. et al, Acta Crystallogr., Sect . D: Biol. Crystallogr. 2004, D60, 2126-2132] for refinement. See Table 8 for final model statistics. Table 8 : Data collection and refinement statistics obtained from Fab ticagrelor co-crystal structures Values in parentheses refer to the highest resolution shell. describe TICA0072 TICA0212 (MEDI2452)
PDB access code
data collection statistics
radiation source ESRF/ID23-1 ESRF/ID23-1
radiation detector Pilatus Pilatus
space group P21 P21212
Cell size a=41.2, b=72.6, c=67.8 β=98.9 a=69, b=173, c=42
Resolution (Å) 49-1.7 (1.87-1.7) 41-2.16 (2.27-2.16)
reflection of observation 143002 232480
unique reflection 41888 36570
Integrity (%) 97.0 (95.3) 99.4 (94.7)
Mean I/σ I 13.6(1.7) 10.7(1.2)
R sym % b 4.8 (74.4) 9.7(27.7)
Refinement Statistics
Resolution (Å) 49-1.7 41-2.16
Protein + Ligand Number of Atoms 3308 3371
number of solvent atoms 142 87
R (%), R free (%) 19.8, 23.7 21.7, 25.7
Wilson B(Å 2 ), Refinement <B>(Å 2 ) 45.6, 48.5 42.1, 44.6
Rmsd ideal bond length (Å) 0.008 0.010
Bond angle (°) 1.10 1.25
The structure of TICA0072 in complex with ticagrelor was determined at 1.7 Å resolution (Figure 10). The CDRs form highly concave surfaces and ticagrelor is deeply inserted into the interface between the VH and VL domains. This type of binding is typically observed in small haptens. All CDRs except VL CDR2 directly constitute ticagrelor binding and most VH CDR3 abnormalities. The difluorophenyl group of ticagrelor is located in a cavity filled with hydrophobic residues including the vernier residues VH Trp47, VL Phe98 and the VH CDR3 residue Leu100L. The major residue that interacts with ticagrelor is VL Trp91, which is involved in pi-stacking against the adenosine-like core and hydrogen bonding to one of the ribose hydroxyl groups of the cyclopentyl moiety. Additional interactions with the adenosine-like core are provided by VH CDR1 His35 and VH CDR3 Tyr99. Thiopropyl substituents stack against the backbone of the VH CDR2 loop. The hydroxyethyl substituent on the cyclopentyl moiety extends into the solvent and does not produce any interaction with the Fab.
In the structure of the affinity-improved Fab TICA0212/MEDI2452, ticagrelor binding was similar to ticagrelor binding of TICA0072 with all of the above-mentioned cross retentions but with some important differences (Figure 10B). The combination of VL CDR3 mutations Asp92Leu and Ser94Asp disrupts hydrogen bonds in the VL CDR3 loop to produce a more "relaxed" structure. The new configuration is associated with a pyrimidine ring tilted at 15° with respect to the attached cyclopropyl-difluorophenyl substituent. The new position of the pyrimidine ring makes it about 0.2 Å closer to VH CDE3Tyr99 in TICA0212/MEDI2452 than Fab72. In addition, VL Ile93Tyr introduces a hydrogen bond donor that creates interaction with the VH CDR3 loop, thereby defining further binding sites.
The crystal structure of TICA0212/MEDI2452 shows that ticagrelor is restricted to VH
with VL
Deep fissures between interfaces. Since the crystal structure indicated that the hydroxyethyl group was not involved in any interaction with TICA0212/MEDI2452, the design strategy of using biotin to label ticagrelor via a triamide linker to the hydroxyethyl group was confirmed. This is further supported by the fact that Fab also binds TAM lacking hydroxyethyl groups with the same affinity. TICA0212/MEDI2452 showed weak binding to TIM lacking cyclopropyl-difluorophenyl. In the TICA0212/MEDI2452-ticagrelor complex, the cyclopropyl-difluorophenyl is buried at the bottom of the hydrophobic cavity and must have V in alignmentL
The CDR3 residue Trp L91 plays a key structural role in the ticagrelor-like adenosine core. Since the chemical starting point of ticagrelor is ATP and its retaining adenosine-like core, a key attribute of the antidote's specificity is the demonstration that it does not bind adenosine. This pilot isolation strategy includes high yield and detailed specificity assays and detection of unbound adenosine by competitive or direct binding assays. From structural analysis, it is expected that the purine ring and ribosyl group of adenosine mimic the interaction of an adenosine core like ticagrelor. However, the lack of binding can be explained by the absence of two hydrophobic R groups (cyclopropyl-difluorophenyl and thiopropyl) that significantly reduce the hydrophobicity of relative shape complementarity and binding interactions.
Structural analysis of the parental TICA0072 and TICA0212/MEDI2452 showed the importance of introducing some changes during affinity maturation. at VL
Mutations in CDR3 were shown to have a particularly significant effect, resulting in different loop configurations and additional hydrogen bonds in TICA0212/MEDI2452 to define the binding pocket. In contrast, at VH
The effect of mutations in CDR3 is less pronounced. These observations from the structure are in part made by containing only VL
CDR3 (TICA0152) or VH
Data for the modified antibody modified in CDR3 (TICA0162) confirmed that it resulted in a 200-fold and 50-fold improvement, respectively, compared to TICA0072. However, although VL
CDR3 alterations were shown to have a higher impact, with significant improvement in the addition of two sets of mutations. It should be noted that the crystal structure is a still picture of the complex and cannot capture any protein and ligand dynamics involved in binding.example 9 : in ticagrelor or TAM exists in vitro TICA0212/MEDI2452 Concentration-dependent recovery of platelet aggregation
The extent and ability of TICA0212/MEDI2452 to reverse ticagrelor- or TAM-mediated inhibition of ADP-induced platelet aggregation was determined in human platelet-rich plasma (PRP) using a light transmittance aggregometry.
For in vitro human PRP analysis, blood was collected by cephalic venipuncture from fasting healthy volunteers. Discard the initial 2 mL of blood, 1+9 (citrate + blood), to a final concentration of 10.9 mM before collecting aliquots into tubes containing 0.109 M sodium citrate. Anticoagulated human blood was centrifuged at 240 x g for 15 min. Carefully remove PRP and transfer to a clean vial. Platelet poor plasma (PPP) was prepared by centrifuging PRP at 2000 x g for 15 min. Light transmittance agglutination (LTA) was assessed in PRP by a platelet aggregation mapper (PAP-8E, Bio/Data Corporation, PA, USA). Zero% agglomeration is defined as the light transmission of the PRP and 100% agglomeration is defined as the light transmission of the PPP.
PRP was pre-incubated with 1 μM ticagrelor or TAM for 1 hour prior to co-incubation with different concentrations of TICA0212 or isotype control Fab for 30 minutes. Platelet aggregation was initiated by addition of 20 μM ADP and recording continued for 6 min. The final aggregation (FA) degree data at 6 min was analyzed.
Calculate the concentration of TICA0212/MEDI2452 that provides half-maximal reversal (IC50
). TICA0212/MEDI2452 produced a concentration-dependent reversal of 1 μM ticagrelor and 1 μM TAM-mediated inhibition of 20 μM ADP-induced platelet aggregation, calculated mean (n=5) IC50
values were 0.64 and 0.78 μM, respectively (picture 8
). When assessed in 1:1 conditions (1 μM TICA0212/MEDI2452:1 μM ticagrelor or TAM), the mean degree of reversal was 78% and 62%, respectively. The isotype control Fab did not induce significant reversal of ticagrelor and TAM ADP-induced platelet aggregation. For example, isotype control Fab produced -3% and 2% ticagrelor and TAM reversal, respectively, after 30 min incubation.
Thus, the data show that TICA0212/MEDI2452 can reverse ticagrelor- and TAM-mediated inhibition of ADP-induced platelet aggregation in vitro in a concentration-dependent manner. A maximal and nearly complete reversal effect was achieved when evaluated in a 1:1 experimental setting (predictably when TICA0212/MEDI2452 was bound to ticagrelor or TAM in a 1:1 stoichiometric ratio).example 10 : in ticagrelor or TAM exist TICA0212/MEDI2452 Effective and rapid recovery of platelet aggregation in vitro
The onset time of TICA0212/MEDI2452 reversal of ticagrelor or TAM was determined in human platelet-rich plasma (PRP) using a light transmission aggregometry as in Example 8. PRP was pre-incubated with 1 μM ticagrelor or TAM for 1 hour before addition of 1 μM TICA0212/MEDI2452 and co-incubation for 5, 10, 15, 30, and 60 minutes, or addition of isotype control Fab for 30 minutes. Platelet aggregation was initiated by addition of 20 μM ADP and recording continued for 6 min. The final aggregation (FA) degree data at 6 min was analyzed.
TICA0212/MEDI2452 produced similar degrees of reversal of ticagrelor-mediated inhibition regardless of co-culture time, with mean (n=3) degrees of reversal of 85% and 69% after 5, 10, 15, 30 and 60 minutes, respectively , 74%, 80%, and 81%. Similarly, the mean (n=3) degree of TAM reversal induced by TICA0212/MEDI2452 after 5, 10, 15, 30 and 60 minutes was 53%, 56%, 58%, 69%, 74%, respectively. TICA0212/MEDI2452 rapidly and efficiently reversed ticagrelor and mediated ADP-induced inhibition of platelet aggregation. There was no reversal (mean (n=3)-2%) after 30 min of co-incubation with the isotype control Fab. Based on this data, TICA0212/MEDI2452 showed rapid and potent reversal of ticagrelor- and TAM-mediated inhibition of ADP-induced aggregation when evaluated in a 1:1 experimental setting.Example 11 : after administration to ticagrelor-treated mice TICA0212/MEDI2452 Effective and rapid recovery of platelet aggregation in vivo
In vitro determination of TICA0212/MEDI2452-mediated reversal of ticagrelor-mediated inhibition of ADP-induced platelet aggregation in whole blood (impedance aggregometry) following intravenous (i.v.) administration of ticagrelor to mice The speed and extent of onset of action. Ticagrelor was administered intravenously to mice at a bolus of 1200 μg/kg over 5 minutes, followed by a continuous infusion of 30 μg/kg/min over 15 minutes. Following termination of ticagrelor infusion, mice were administered an intravenous bolus of 250 mg/kg TICA0212/MEDI2452 when the measured plasma exposure of ticagrelor was an average of 1.4 μM. After 5, 30, and 60 min of TICA0212/MEDI2452 administration, mice were sacrificed and blood samples were collected. ADP-induced agglutination responses were measured for 6 min in this study and data were expressed as the mean area under the curve of agglutination units (AU) recorded over time (AU*min).
For impedance aggregometry analysis, mice were killed and blood samples collected to 7 μM hirudin. Blood (175 μL) was added to pre-warmed NaCl in a multiplate microtest unit2
(37°C, 175 μL) and mixed for 3 min before adding 12 μL ADP to a final concentration of 6.5 μM. The disposable multi-plate microtest unit contains a stir bar and has two separate pairs of electrodes immersed in the blood sample.
When an agonist (ADP) was added and shear induced by agitation, platelets began to attach and aggregate on the electrodes. This resulted in an increase in impedance on the electrodes, which was continuously recorded over time by a multi-plate impedance aggregometer (DynaByte, Munchen, Germany).
As the mean (n=4) aggregation response decreased from 432 to 2, from 474 to 6, and from 494 to 14 AU*min (5, 30 and 60 min after ticagrelor infusion and PBS bolus, respectively).picture 9
), so ticagrelor treatment induced almost complete inhibition of ADP-induced aggregation. As the mean (n=4) coagulation response increased from 2 to 147, from 6 to 448, and from 14 to 412 AU*min (picture 9
), so TICA0212/MEDI2452-mediated reversal of ticagrelor-mediated inhibition in vivo. As the mean (n=4) coagulation reaction remained unchanged from 6 to 4 AU*min, there was no reversal 30 minutes after administration of the isotype control.
This data shows that TICA0212/MEDI2452 can rapidly and effectively restore ADP-induced platelet aggregation when administered intravenously in high doses to mice administered at a plasma concentration of 1.4 μM ticagrelor, which provides complete inhibition of ADP-induced aggregation.Example 12 : Bleeding in mice treated with ticagrelor
Since one of the expected indications for TICA0212/MEDI2452 is as an antidote for ticagrelor patients requiring emergency surgery, TICA0212/MEDI2452 was evaluated in a mouse bleeding assay designed to mimic a clinical setting that should achieve complete reversal before surgery begins MEDI2452.
Mice were pretreated via continuous infusion of ticagrelor (300 μg/kg/min) or vehicle for 20 minutes. After cessation of infusion, t=0, a bolus of TICA0212/MEDI2452 (600 mg/kg) or vehicle (histidine-sucrose buffer) was administered over 45 sec and at t=30 min by severing the 5 mm tail tip induced bleeding. The tail tip was rinsed with water (2 mL/min) and the blood and water mixture was collected in a small vessel where the liquid was mixed with agitation to enhance hemolysis and establish a homogeneous solution. Light transmission at 525 nm for 30 minutes was recorded for platelet aggregation when terminal blood samples were collected from the abdominal aorta. Light transmittance can be converted to absorbance and used to calculate blood loss as area under the absorption curve (AUC, absorbance*s) and total bleeding time (BT, s) by plotting absorbance over time. All transmittances below 95% were defined as bleeding. Platelet aggregation and total and free plasma exposure samples were also collected at the end of ticagrelor infusion (t=0) and at tail severing (t=30 min).
The study was approved by the Animal Research Ethics Committee of the University of Göteborg, Sweden. Mice were anesthetized using isoflurane gas (Forene®, Abbot Scandinavia AB, Sweden). The left jugular vein is cannulated for administration of vehicle or drug. Body temperature was maintained at 38°C by external heat.
To translate the effect of TICA0212/MEDI2452 on platelet aggregation into a potential hemorrhagic effect, a mouse tail hemorrhage prevention study was performed. Ticagrelor was fused to mean overall Grelor and TAM plasma concentrations of 7.6 and 0.3 μM, respectively, to provide a significant drug-dependent bleeding window. A single bolus dose of 600 mg/kg TICA0212/MEDI2452 was administered over 30 seconds immediately after discontinuation of the ticagrelor infusion. After 30 minutes, ADP-induced aggregation was fully normalized and the mean free plasma concentration of ticagrelor decreased from 4.7 nM to below 0.03 nM (lower limit of quantification). Bleeding was initiated by tail severing and monitored for 30 minutes. In vehicle-treated mice, mean total ticagrelor and TAM plasma concentrations were 2.4 and 0.6 μM at tail severance. Total blood loss and bleeding time over 30 minutes were significantly (p<0.05) enhanced by ticagrelor about 3.8-fold and 1.6-fold, respectively. TICA0212/MEDI2452 significantly (p<0.05) reversed blood loss and bleeding time relative to ticagrelor alone and recovered to an extent that was not significantly different from ticagrelor-untreated mice (Figure 12). In this prophylactic setting, TICA0212/MEDI2452 normalized ticagrelor-dependent bleeding. The 30-minute onset time transferred from the in vitro model to this in vivo model demonstrated that TICA0212/MEDI2452 restored blood loss and bleeding time to those of ticagrelor-untreated mice.Example 13 : Ticagrelor and TAM total plasma concentration
Blood samples were collected in tubes with EDTA anticoagulant and centrifuged at 10000 x g for 5 min at room temperature to prepare plasma. Plasma concentrations of ticagrelor and TAM were determined by protein precipitation and liquid chromatography using tandem mass spectrometry (LC-MS/MS), as in published methods with the following deviations [Sillén H. et al., J. Chromatogr B Analyt Technol Biomed Life Sci 2010;878:2299–306]. Plasma (50 μL) was subjected to protein precipitation in acetonitrile using 180 μl internal standard (D7-ZD6140). The liquid chromatography system and mass spectrometer were obtained from an Acquity Ultra Performance LC coupled Xevo TQ-S mass spectrometer from Waters. Chromatographic separation was achieved on an Aquity UPLC® BEH C18 column (2.1 x 50 mm, particle size 1.7 µm). Negative electrospray ionization was used. Eluent A is water containing 10 mmol/L ammonium acetate, pH 5 and eluent B is acetonitrile containing 10 mmol/L ammonium acetate. Injection volumes ranged from 1 to 5 μL and the analytical gradient started at 4%B, increased to 95% in 1.5 min, maintained until 2.3 min, then returned to initial conditions in 2.4 min, followed by equilibration again for 0.3 min. No quality control samples were used in this analysis. The lower limit of quantification (LLOQ) was 0.005 μmol/L and the calibration range was 0.005 to 15.0 μM.
Total plasma concentrations of ticagrelor and TAM in the presence of TICA0212/MEDI2452 were determined by addition of 1% formic acid (FA:sample, 1:5) followed by protein precipitation and LC-MS/MS as described above. Formic acid was added to the samples to facilitate dissociation of ticagrelor and TICA0212/MEDI2452.example 14 : Free plasma concentration of ticagrelor
The method was optimized based on a previously published method [Sillén H. et al, J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Aug 1;879(23):2315-22]. Dialysis membranes (Spectrum Laboratories, Inc) that allow passage of molecules with masses below 6 to 8 kDa were soaked in ELGA water for 10 to 15 min and placed between semidialysis plates (in-house prepared). Add 130 μL of plasma to one side of the dialysis plate and 130 μL of phosphate buffer (pH 7.0) to the opposite side. Cover the holes on both sides with lids and place an aluminum plate on top of each side of the plate to avoid leakage. The plate was then placed vertically on a rotary shaker at 100 rpm/min for 24 h at 37°C. Dialysis was terminated by transferring 50 μL of retentate from the plasma side and 75 μL of dialysate from the buffer side to a protein LoBind PCR clean 96 deep well plate containing 150 μL of each 75 μL internal standard in acetonitrile (D7-ZD6140). The plate was mixed for 1 min and then centrifuged at 1500 x g for 20 min at 4°C. After centrifugation, transfer 50 μL of the supernatant obtained from the pellet retentate and use 50 μL of ELGA H2
O dilution followed by LC-MS/MS (Acquity Ultra Performance LC coupled to a Xevo TQ-S mass spectrometer, Waters) analysis. No quality control samples were used in this analysis. The retentate calibration range was 0.4 to 1000 nmol/L and the dialysate calibration range was 0.003 to 50 nmol/L. The LLOQ of ticagrelor in dialysate was 0.03 nmol/L.
Table 9 below provides a summary of the exemplary antibodies described in the examples above and serves to illustrate certain embodiments of the techniques disclosed herein.
Table 9. Summary of antibody/scFv/Fab sequences Antibody Ref sequence sequence
TICA0010 Vh DNA (SEQ ID NO: 1) gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc aacagagtac gacctgcaac ggcctttcgg gtttgacttc tggggcaagg ggacaatggt caccgtctcg agt
TICA0010 Vh (SEQ ID NO:2) EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAT EYDLQRPFGFDFWGKGTMVTVSS
TICA0010 Vh CDR1 (SEQ ID NO:3) SYAMS
TICA0010 Vh CDR2 (SEQ ID NO:4) AISGSGGSTYYADSVKG
TICA0010 Vh CDR3 (SEQ ID NO:5) EYDLQRPFGFDF
TICA0010 Vl DNA (SEQ ID NO:6) tcctatgtgc tgactcagcc accctcagcg tctggggccc ccgggcagag ggctaccatc tcctgctctg gaagcagctc caacatcgga agtaatcttg tgaactggta ccaacaattc ccaggagagg cccccaagct cctcatcttt agtgacaatc aacgaccctc aggggtccct gaccgattct ctggctccag gtctggcacc tcagcctccc tggccatcag tgggctccag tccgaggatg aggctgatta ttactgtgca acgtgggatg acagactgga tggttatgtg gtattcggcg gagggaccaa gctgaccgtc cta
TICA0010 Vl (SEQ ID NO:7) SYVLTQPPSASGAPGQRATISC SGSSSNIGSNLVN WYQQFPGEAPKLLIF SDNQRPS GVPDRFSGSRSGTSASLAISGLQSEDEADYYC ATWDDRLDGYVVFGGGTKLTVL
TICA0010 V1 CDR1 (SEQ ID NO:8) SGSSSNIGSNLVN
TICA0010 V1 CDR2 (SEQ ID NO:9) SDNQRPS
TICA0010 Vl CDR3 (SEQ ID NO: 10) ATWDDRLDGYVV
TICA0049 Vh DNA (SEQ ID NO: 11) caggtacagc tgcagcagtc aggggctgag gtgaagaagc ctggggcctc agtgaaggtt tcctgcaagg cttctggata caccttcatt acctatggta ttcactgggt gcgccaggcc cccggacaag ggcttgagtg gatgggatgg atcgaccccg ggcatggtta cacaaaatat tcacagaagt tccagggcag agtcaccatt accagggaca catccgcgag cacagcctac atggagatga gcagcctcag atctgaagac acggctgtgt attactgtgc gagagcggac ctgggtgact actggggccg gggaaccctg gtcaccgtct cgagt
TICA0049 Vh (SEQ ID NO: 12) QVQLQQSGAEVKKPGASVKVSCKASGYTFI TYGIH WVRQAPGQGLEWMG WIDPGHGYTKYSQKFQG RVTITRDTSASSTAYMEMSSLRSEDTAVYYCAR ADLGDY WGRGTLVTVSS
TICA0049 Vh CDR1 (SEQ ID NO: 13) TYGIH
TICA0049 Vh CDR2 (SEQ ID NO: 14) WIDPGHGYTKYSQKFQG
TICA0049 Vh CDR3 (SEQ ID NO: 15) ADLGDY
TICA0049 Vl DNA (SEQ ID NO: 16) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcagctc caacattggg aagaattatg tttcctggtt ccagcagctc ccaggtacag cccccaaact cctcatttat gacaatcata agcgaccctc agggattcct gaccgattct ctgcctccaa gtctggcacg tcagccaccc tggtcatctc cggtctccag actggggacg aggcccatta ttactgcgga acatgggata ccagactgag tgctggggtg ttcggcggag ggaccaaggt caccgtccta
TICA0049 Vl (SEQ ID NO: 17) QSVVTQPPSVSAAPGQKVTISCSGSSSNIGKNYVSWFQQLPGTAPKLLIYDNHKRPSGIPDRFSASKSGTSATLVISGLQTGDEAHYYCGTWDTRLSAGVFGGGTKVTVL _ _
TICA0049 V1 CDR1 (SEQ ID NO: 18) SGSSSNIGKNYVS
TICA0049 V1 CDR2 (SEQ ID NO: 19) DNHKRPS
TICA0049 Vl CDR3 (SEQ ID NO:20) GTWDTRLSAGV
TICA0053 Vh DNA (SEQ ID NO:21) gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgg ccatgatagt agtggttact cctactcctt tgacttctgg gggcggggga ccacggtcac cgtctcgagt
TICA0053 Vh (SEQ ID NO: 22) EVQLLESGGGLVQPGGSLRLSCAASGFTFS SYAMS WVRQAPGKGLEWVS AISGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCGH DSSGYSYSFDF WGRGTTVTVSS
TICA0053 Vh CDR1 (SEQ ID NO: 23) SYAMS
TICA0053 Vh CDR2 (SEQ ID NO: 24) AISGSGGSTYYADSVKG
TICA0053 Vh CDR3 (SEQ ID NO:25) DSSGYSYSFDF
TICA0053 Vl DNA (SEQ ID NO:26) cagtctgtgt tgacgcagcc gccctcagcg tctgggaccc ccgggcagag ggtcaccatc tcttgttctg gcaacatctc caacatcgga agtaacactg tcaactggta tcaacacgtc ccaggagcgg cccccagact cctcatctat gttaatgatc agcggccgtc aggggtccct gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag tctgaagatg aggctgatta ttactgtgca acgtgggatg acaccctgaa tggaggggtc ttcggcggag ggaccaagct gaccgtccta
TICA0053 Vl (SEQ ID NO:27) QSVLTQPPSASGTPGQRVTISC SGNISNIGSNTVN WYQHVPGAAPRLLIY VNDQRPS GVPDRFSGSKSGTSASLAISGLQSEDEADYYC ATWDDTLNGGVFGGGTKLTVL
TICA0053 V1 CDR1 (SEQ ID NO:28) SGNISNIGSNTVN
TICA0053 V1 CDR2 (SEQ ID NO:29) VNDQRPS
TICA0053 Vl CDR3 (SEQ ID NO:30) ATWDDTLNGGV
TICA0072 Vh DNA (SEQ ID NO:31) caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagagggtcc catctttacg atttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc caaggaaccc tggtcaccgt ctcgagt
TICA0072 Vh (SEQ ID NO:32) QVQLQESGAEVKKPGSSVRVSCKASGGTFD SYSIH WVRQAPGQGLEWMG GIIPAFGTLSSAQDFQA RVTISADKSTSTAYMELSGLRSEDTAVYYCAR GSHLYDFWSASHPPNDALAIWGQGTLVTVSS
TICA0072 Vh CDR1 (SEQ ID NO:33) SYSIH
TICA0072 Vh CDR2 (SEQ ID NO:34) GIIPAFGTLSSAQDFQA
TICA0072 Vh CDR3 (SEQ ID NO:35) GSHLYDFWSASHPPNDALAI
TICA0072 Vl DNA (SEQ ID NO:36) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag gctggggacg aggccgatta ttactgcggg acatgggata tcagcctgag cgctggcttg ttcggcggag ggaccaaggt caccgtccta
TICA0072 V1 (SEQ ID NO:37) QSVVTQPPSVSAAPGQKVTISC SGSNSDIGNNYVS WYQQLPGTAPKLLIY DNNKRPS GIPDRFSGSKSGTSATLAITGLQAGDEADYYC GTWDISLSAGLFGGGTKVTVL
TICA0072 V1 CDR1 (SEQ ID NO:38) SGSNSDIGNNYVS
TICA0072 V1 CDR2 (SEQ ID NO:39) DNNKRPS
TICA0072 V1 CDR3 (SEQ ID NO:40) GTWDISLSAGL
TICA0159 Vh DNA (SEQ ID NO:41) caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagagggagc ttcgactaca ggttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc caaggaaccc tggtcaccgt ctcgagt
TICA0159 Vh (SEQ ID NO:42) QVQLQESGAEVKKPGSSVRVSCKASGGTFD SYSIH WVRQAPGQGLEWMG GIIPAFGTLSSAQDFQA RVTISADKSTSTAYMELSGLRSEDTAVYYCAR GSFDYRFWSASHPPNDALAI WGQGTLVTVSS
TICA0159 Vh CDR1 (SEQ ID NO:43) SYSIH
TICA0159 Vh CDR2 (SEQ ID NO:44) GIIPAFGTLSSAQDFQA
TICA0159 Vh CDR3 (SEQ ID NO:45) GSFDYRFWSASHPPNDALAI
TICA0159 Vl DNA (SEQ ID NO:46) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag gctggggacg aggccgatta ttactgcggg acatgggata tcagcctgag cgctggcttg ttcggcggag ggaccaaggt caccgtccta
TICA0159 Vl (SEQ ID NO:47) QSVVTQPPSVSAAPGQKVTISC SGSNSDIGNNYVS WYQQLPGTAPKLLIY DNNKRPS GIPDRFSGSKSGTSATLAITGLQAGDEADYYC GTWDISLSAGLFGGGTKVTVL
TICA0159 V1 CDR1 (SEQ ID NO:48) SGSNSDIGNNYVS
TICA0159 V1 CDR2 (SEQ ID NO:49) DNNKRPS
TICA0159 V1 CDR3 (SEQ ID NO:50) GTWDISLSAGL
TICA0162 Vh DNA (SEQ ID NO:51) caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagaggctcc ttcgactact acttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc caaggaaccc tggtcaccgt ctcgagt
TICA0162 Vh (SEQ ID NO:52) QVQLQESGAEVKKPGSSVRVSCKASGGTFD SYSIH WVRQAPGQGLEWMG GIIPAFGTLSSAQDFQA RVTISADKSTSTAYMELSGLRSEDTAVYYCAR GSFDYYFWSASHPPNDALAIWGQGTLVTVSS
TICA0162 Vh CDR1 (SEQ ID NO:53) SYSIH
TICA0162 Vh CDR2 (SEQ ID NO:54) GIIPAFGTLSSAQDFQA
TICA0162 Vh CDR3 (SEQ ID NO:55) GSFDYYFWSASHPPNDALAI
TICA0162 Vl DNA (SEQ ID NO:56) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag gctggggacg aggccgatta ttactgcggg acatgggata tcagcctgag cgctggcttg ttcggcggag ggaccaaggt caccgtccta
TICA0162 Vl (SEQ ID NO:57) QSVVTQPPSVSAAPGQKVTISC SGSNSDIGNNYVS WYQQLPGTAPKLLIY DNNKRPS GIPDRFSGSKSGTSATLAITGLQAGDEADYYC GTWDISLSAGLFGGGTKVTVL
TICA0162 V1 CDR1 (SEQ ID NO:58) SGSNSDIGNNYVS
TICA0162 V1 CDR2 (SEQ ID NO:59) DNNKRPS
TICA0162 V1 CDR3 (SEQ ID NO:60) GTWDISLSAGL
TICA0152 Vh DNA (SEQ ID NO:61) caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagagggtcc catctttacg atttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc caaggaaccc tggtcaccgt ctcgagt
TICA0152 Vh (SEQ ID NO:62) QVQLQESGAEVKKPGSSVRVSCKASGGTFD SYSIH WVRQAPGQGLEWMG GIIPAFGTLSSAQDFQA RVTISADKSTSTAYMELSGLRSEDTAVYYCAR GSHLYDFWSASHPPNDALAIWGQGTLVTVSS
TICA0152 Vh CDR1 (SEQ ID NO:63) SYSIH
TICA0152 Vh CDR2 (SEQ ID NO:64) GIIPAFGTLSSAQDFQA
TICA0152 Vh CDR3 (SEQ ID NO:65) GSHLYDFWSASHPPNDALAI
TICA0152 Vl DNA (SEQ ID NO:66) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag gctggggacg aggccgatta ttactgcggg acatggctgt acgaccgggc cgtcggcttg ttcggcggag ggaccaaggt caccgtccta
TICA0152 Vl (SEQ ID NO:67) QSVVTQPPSVSAAPGQKVTISC SGSNSDIGNNYVS WYQQLPGTAPKLLIY DNNKRPS GIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWLYDRAVGLFGGGTKVTVL
TICA0152 V1 CDR1 (SEQ ID NO:68) SGSNSDIGNNYVS
TICA0152 V1 CDR2 (SEQ ID NO:69) DNNKRPS
TICA0152 V1 CDR3 (SEQ ID NO:70) GTWLYDRAVGL
TICA0212/MEDI2452 Vh DNA (SEQ ID NO:71) caggtgcagc tgcaggagtc cggggctgag gtgaagaagc ctgggtcctc ggtgagggtc tcctgcaagg cttctggagg caccttcgac agttatagta tccattgggt gcgccaggcc cctggacaag ggcttgagtg gatgggaggg atcatccctg cctttgggac attaagcagc gcacaggact tccaggccag agtcaccatt agcgcggaca agtccacgag cacagcctat atggagctga gcggcctgag atctgaggac acggccgtat attactgtgc gagaggctcc ttcgactact acttttggag tgcttctcat ccccccaatg atgctcttgc tatttggggc caaggaaccc tggtcaccgt ctcgagt
TICA0212/MEDI2452 Vh (SEQ ID NO:72) QVQLQESGAEVKKPGSSVRVSCKASGGTFD SYSIH WVRQAPGQGLEWMG GIIPAFGTLSSAQDFQA RVTISADKSTSTAYMELSGLRSEDTAVYYCAR GSFDYYFWSASHPPNDALAIWGQGTLVTVSS
TICA0212/MEDI2452 Vh CDR1 (SEQ ID NO:73) SYSIH
TICA0212/MEDI2452 Vh CDR2 (SEQ ID NO:74) GIIPAFGTLSSAQDFQA
TICA0212/MEDI2452 Vh CDR3 (SEQ ID NO:75) GSFDYYFWSASHPPNDALAI
TICA0212/MEDI2452 Vl DNA (SEQ ID NO:76) cagtctgtcg tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc tcctgctctg gaagcaactc cgacattggc aacaattatg tgtcgtggta ccaacagctc ccaggaacag cccccaaact cctcatttat gacaataata aacgaccctc agggattcct gaccgattct ctggctccaa gtctggcacg tcagccaccc tggccatcac cggactccag gctggggacg aggccgatta ttactgcggg acatggctgt acgaccgggc cgtcggcttg ttcggcggag ggaccaaggt caccgtccta
TICA0212/MEDI2452 Vl (SEQ ID NO:77) QSVVTQPPSVSAAPGQKVTISC SGSNSDIGNNYVS WYQQLPGTAPKLLIY DNNKRPS GIPDRFSGSKSGTSATLAITGLQAGDEADYYCGTWLYDRAVGLFGGGTKVTVL
TICA0212/MEDI2452 V1 CDR1 (SEQ ID NO:78) SGSNSDIGNNYVS
TICA0212/MEDI2452 V1 CDR2 (SEQ ID NO:79) DNNKRPS
TICA0212/MEDI2452 Vl CDR3 (SEQ ID NO:80) GTWLYDRAVGL
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All publications and patent applications mentioned herein are incorporated by reference in their entirety to the same extent as if individual publications or patent applications were specifically and individually incorporated by reference.
The foregoing description is illustrative and not restrictive when discussing particular aspects of the invention. Numerous variations of the present invention will become apparent to those skilled in the art upon examination of this specification and the scope of the following claims. The full scope of the present invention should be determined by reference to the full scope of the claims and their equivalents, and the specification, and such variations.
